Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Writing Revision List

Line 1. Should be written: Short-Lived Exercise-Induced Exerkines Modulate Inflammation for Chronic Disease Prevention: A Systematic Review and Meta-Analysis
Line 5. Should be written: ¹,⁴, Bakhtyar Tartibian¹, Seyed Morteza Tayebi¹, and Rahman Sheikhhoseini³
Line 10. Should be written: Physical exercise triggers short-lived exerkines, such as interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10), which may help reduce systemic inflammation and mitigate the risk of chronic disease.
Line 11. Should be written: Despite their potential, the effects of these exerkines across diverse populations remain underexplored.
Line 14. Should be written: We systematically searched PubMed, Scopus, and Web of Science from January 2015 to February 2025, identifying 11 randomized controlled trials (RCTs) involving 1,135 participants.
Line 19. Should be written: Heterogeneity was moderate for CRP (I² = 52.5%) but high for other markers (I² > 87%).
Line 20. Should be written: These findings suggest that exerkines foster an anti-inflammatory environment, supporting their role in preventing cardiometabolic diseases; however, standardised protocols and longitudinal studies are needed to confirm long-term benefits.
Line 26. Should be written: Among these, the release of exercise-induced cytokines, often termed "exerkines," has emerged as a critical mediator linking acute physical activity to systemic health outcomes [2].
Line 30. Should be written: Although these cytokines typically peak within hours of exercise and return to baseline soon after, growing evidence suggests that such transient responses may trigger cumulative adaptations, potentially offering long-term protection against chronic diseases such as cardiovascular disease, type 2 diabetes, and obesity [4, 5].
Line 33. Should be written: Notably, transient exerkine elevations may drive prolonged benefits by modulating immune responses, a process central to their protective role.
Line 43. Should be written: Despite compelling evidence linking exerkines to acute physiological changes, significant gaps remain in our understanding of their broader implications.
Line 44. Should be written: Most research has examined the immediate effects in trained athletes or clinical populations, leaving the impact on healthy adults and those with or at risk for chronic conditions largely unexplored [12, 13].
Line 48. Should be written: Moreover, although systematic reviews have explored myokines in the context of disease management or broader metabolic effects, few have specifically addressed the capacity of short-lived exerkines to contribute to long-term health outcomes in healthy individuals [16, 17].
Line 59. Should be written: The primary aim of this systematic review and meta-analysis was to evaluate the overall impact of exercise-induced exerkines on long-term disease prevention and management in adults, including both healthy individuals and those with specific chronic conditions or elevated disease risk.
Line 63. Should be written: interventions, including aerobic, resistance, and high-intensity interval training.
Line 77. Should be written: The keywords (“exercise” OR “physical activity” OR “aerobic exercise” OR “resistance training” OR “high-intensity interval training” OR “exercise training”) AND (“cytokine” OR “cytokines” OR “pro-inflammatory cytokines” OR “anti-inflammatory cytokines” OR “exerkine” OR “exerkines”) AND (“disease prevention” OR “chronic disease” OR “long-term health” OR “health outcomes”) were used to conduct the searches.
Line 87. Should be written: For the population, studies included adults aged 18 years or older, comprising healthy individuals, those diagnosed with specific chronic conditions (e.g., sarcopenia, rheumatoid arthritis, cognitive impairment), or those at elevated risk of chronic diseases (e.g., individuals with heightened breast cancer susceptibility).
Line 90. Should be written: Comparison groups consisted of non-exercising or inactive controls who maintained their habitual lifestyle without structured interventions.
Line 93. Should be written: Outcomes encompassed primary measures of short-term alterations in circulating exerkine concentrations (e.g., IL-6, TNF-alpha, IL-10, CRP, IFN-gamma) assessed pre- and post-intervention, and secondary endpoints related to chronic disease prevention or management, including systemic inflammation, metabolic parameters, and cardiovascular performance.
Line 100. Should be written: Studies were excluded if they involved animal models or in vitro experiments, lacked a clearly defined control group, or included confounding interventions (e.g., dietary or pharmacological treatments).
Line 104. Should be written: A customised data extraction template was developed and piloted in Microsoft Excel to systematically capture pertinent information from the included studies.
Line 108. Should be written: Data extraction was conducted independently by the primary reviewer (H.P.), with the following variables recorded: (1) study identification (first author, publication year, country); (2) study design (e.g., RCT, parallel or crossover); (3) participant characteristics (sample size, age, sex, health status); (4) intervention details (exercise type, duration, frequency, intensity, and mode of delivery); (5) comparator details (non-exercising or inactive control group); (6) outcome measures, including primary outcomes (pre- and post-intervention concentrations of circulating exerkines such as IL-6, TNF-alpha, IL-10, CRP, IFN-gamma) and secondary outcomes (systemic inflammation markers, metabolic parameters, cardiovascular performance, and adverse events); and (7) key findings.
Line 115. Should be written: For studies presenting data solely in graphical format, numerical values were extracted with high precision using Web Plot Digitizer software [21].
Line 117. Should be written: Studies with unresolved data gaps were excluded from the quantitative synthesis for the affected outcomes.
Line 122. Should be written: Meta-analyses were performed using Comprehensive Meta-Analysis software.
Line 123. Should be written: Standardised mean differences (SMDs) with 95% confidence intervals (CIs) were calculated to evaluate the effects of exercise interventions on circulating exerkine levels, including IL-6, TNF-alpha, IL-10, CRP, and IFN-gamma.
Line 125. Should be written: Heterogeneity was assessed using the I² statistic (I² > 50%: moderate; I² > 75%: high).
Line 126. Should be written: Publication bias was evaluated with funnel plots and Egger's test (P < 0.05 indicating potential bias).
Line 139. Should be written: Participants included healthy postmenopausal women (n=2 studies), individuals with chronic conditions such as rheumatoid arthritis, cognitive impairment, or sarcopenia (n=6), and those at elevated risk of chronic diseases, including obesity, metabolic syndrome, or high breast cancer risk (n=3).
Line 141. Should be written: The total sample size was 1135 participants (1054 females, 81 males), with individual study sizes ranging from 24 to 720 participants.
Line 142. Should be written: Exercise interventions consisted of aerobic training (n=2), resistance training (n=4), combined aerobic and resistance training (n=3), and high-intensity interval training (n=2), with durations ranging from 6 to 52 weeks and frequencies of 2 to 5 sessions per week.
Line 145. Should be written: Primary outcomes focused on circulating exerkines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), C-reactive protein (CRP), and interferon-gamma (IFN-γ).
Line 149. Should be written: Risk of bias for the 11 included RCTs was assessed using the Cochrane Risk of Bias 2 (RoB 2) tool, evaluating five domains: randomization process (D1), deviations from intended interventions (D2), missing outcome data (D3), measurement of the outcome (D4), and selection of the reported result (D5).
Line 152. Should be written: Five studies [28-32] raised some concerns, primarily due to potential bias in D2 (deviations from intended interventions) related to the lack of blinding of participants and personnel.
Line 233. Should be written: A total of three studies were analyzed to assess the impact of exercise interventions on interferon-gamma (IFN-γ) levels [25, 27].
Line 250. Should be written: A total of seven studies were analyzed to assess the impact of exercise interventions on interleukin-6 (IL-6) levels [22, 24, 26, 27, 30, 31].
Line 304. Should be written: This systematic review and meta-analysis, conducted following the PRISMA 2020 guidelines [20], synthesized evidence from 11 randomized controlled trials (RCTs) to investigate the effects of exercise-induced exerkines, specifically C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), on systemic inflammation and their potential role in preventing chronic diseases in adults.
Line 311. Should be written: The findings demonstrate that exercise significantly reduces pro-inflammatory markers, with standardized mean differences (SMDs) of -0.77 (95% CI: -1.20 to -0.33, P = 0.001) for CRP and -1.088 (95% CI: -2.142 to -0.033, P = 0.043) for TNF-α.
Line 315. Should be written: Exercise significantly increased IL-6 levels (SMD = 0.810, 95% CI: 0.095 to 1.526, P = 0.026), while a non-significant trend toward increased IL-10 was observed (SMD = 0.660, 95% CI: -0.088 to 1.408, P = 0.084).
Line 322. Should be written: Sustained reductions in CRP have been linked to a lower incidence of cardiovascular events in longitudinal studies, suggesting that the observed changes may serve as intermediate biomarkers for long-term risk reduction [33].
Line 326. Should be written: The significant increase in IL-6, often misconstrued as solely pro-inflammatory, reflects its context-dependent anti-inflammatory role during exercise, where it stimulates IL-10 production and suppresses TNF-α [2, 8].
Line 329. Should be written: This anti-inflammatory effect of IL-6 is mediated through key signaling pathways, including nuclear factor-kappa B (NF-κB) and Janus kinase/signal transducer and activator of transcription (JAK/STAT).
Line 340. Should be written: IL-6 has been shown to upregulate brain-derived neurotrophic factor (BDNF), promoting neuroplasticity and potentially reducing the risk of cognitive decline [37].
Line 343. Should be written: Enhanced mitochondrial biogenesis, mediated by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), improves cellular energy metabolism and resilience, supporting long-term cardiometabolic health [39].
Line 346. Should be written: The findings of this meta-analysis provide robust evidence for the immunomodulatory effects of exercise, though certain methodological considerations warrant attention.
Line 347. Should be written: Moderate heterogeneity for CRP (I² = 52.5%) and higher heterogeneity for IL-6, IL-10, and TNF-α (I² > 87%) likely reflect variations in exercise modalities (aerobic, resistance, combined, or high-intensity interval training), intervention durations (6 to 52 weeks), and population characteristics, including healthy individuals, those with chronic conditions such as rheumatoid arthritis or sarcopenia, or at-risk groups with obesity or metabolic syndrome.
Line 352. Should be written: In contrast, results for IL-10 and TNF-α showed variability, potentially due to differences in exercise protocols or participant demographics, as observed in studies such as Chagas et al. (2017) (see Supplementary Figures) [22].
Line 354. Should be written: Given the short-term nature of the included interventions, longitudinal studies with clinical outcomes, such as cardiovascular disease or diabetes incidence, are needed to elucidate the long-term preventive potential of exercise-induced exerkines [19].
Line 360. Should be written: Subgroup analyses exploring the impact of exercise type, intensity, or population characteristics could further elucidate sources of heterogeneity and refine effect estimates [20].
Line 366. Should be written: This meta-analysis of 11 RCTs demonstrates that exercise markedly reduces pro-inflammatory markers, such as C-reactive protein and tumour necrosis factor-alpha, while elevating anti-inflammatory interleukin-6 in diverse populations.
Line 369. Should be written: Exerkine-mediated effects position exercise as a vital strategy for mitigating chronic disease risk, particularly for cardiometabolic and inflammatory disorders.
Line 370. Should be written: Exerkines, notably IL-6, show promise as biomarkers for tailoring personalised exercise prescriptions to optimise health outcomes in at-risk groups.

2. Scientific and Stuctural Review List

L. 14, 132. The authors should clarify the search date. The text states the search was conducted on "7 February 2025" and included studies from "January 2015 to February 2025," yet the manuscript is from 2025. This is likely a future date error and should be corrected to the actual search date (e.g., 2023 or 2024).
L. 19, 125, 347. The authors should consistently use "I²" instead of "P" to report heterogeneity statistics (e.g., I² = 52.5%, not P = 52.5%) to avoid confusion with p-values.
L. 63-64. The authors should explicitly state that the meta-analysis was restricted to RCTs, as this is a key inclusion criterion stated later (L. 95) but not in the aims, which mention a broader range of study designs ("randomized controlled trials, observational cohort studies, and experimental designs").
L. 132-133. The authors should report the number of duplicates removed (657 records identified, 463 unique records remained, therefore 194 duplicates removed) for clarity in the PRISMA flow.
L. 141. The authors should acknowledge the significant gender imbalance (1054 females, 81 males) as a potential limitation affecting the generalizability of the findings, particularly to male populations.
L. 234-236. The authors should correct the citation for the IFN-γ analysis. The text cites [25, 27] but the forest plot in Figure 4 lists three studies (Chupel 2017, Despeghel 2021, Furtado 2020). The reference list must be checked and amended accordingly.
L. 250-252. The authors should correct the citation for the IL-6 analysis. The text cites [22, 30] [24, 26, 27, 31], which is an unclear and potentially incomplete format. The forest plot in Figure 5 lists seven studies; the references should be listed completely and accurately (e.g., [22, 24, 26, 27, 30, 31, XX]).
L. 295-297. The authors should ensure the statistics in the Trim and Fill analysis for TNF-α are reported accurately and consistently. The observed SMD is stated as -1.088 earlier (L. 313) and -0.678 here; the 95% CI also appears inconsistent.
L. 307. The authors should consider rephrasing "C-reactive protein (CRP)" as it is not an exerkine (a cytokine released from muscle in response to exercise) but a general inflammatory marker (hepatic acute-phase protein) modulated by exerkines like IL-6. The sentence should clarify that the review investigated the effects of exercise on these inflammatory markers.
L. 347-350. The authors should strengthen the limitations section by explicitly stating that the high heterogeneity observed for most outcomes (I² > 87%) precludes drawing definitive conclusions and limits the robustness of the pooled estimates, as this is a critical interpretation of the statistical findings.
L. 354-355. The authors should expand the future research direction to specifically recommend investigating the dose-response relationship between different exercise modalities (type, intensity, duration) and exerkine responses to help explain the observed heterogeneity.
L. 369. The authors should use the correct term "Exerkines" instead of "Everkines" in the conclusion.

Author Response

Comment 1: Line 1. Should be written: Short-Lived Exercise-Induced Exerkines Modulate Inflammation for Chronic Disease Prevention: A Systematic Review and Meta-Analysis
Response 1: Thank you for your suggestion. We agree with this comment and have revised the title to match the recommended format. The updated title, "Short-Lived Exercise-Induced Exerkines Modulate Inflammation for Chronic Disease Prevention: A Systematic Review and Meta-Analysis," is now presented on page 1, line 2 of the revised manuscript.

 

Comment 2: Line 5. Should be written: ¹,⁴, Bakhtyar Tartibian¹, Seyed Morteza Tayebi¹, and Rahman Sheikhhoseini³
Response 2: Thank you for your feedback. We interpret your suggestion as a request to ensure consistency between the author list and affiliation details. We have revised the author sequence on page 1, line 5 to "HosseinPoorhabibi¹, Rasoul Eslami¹,*, BakhtyarTartibian¹, Seyed Morteza Tayebi¹, Katja Weiss², Thomas Rosemann², BeatKnechtle²,*, and RahmanSheikhhoseini³" to reflect the correct order. Additionally, we have adjusted the email order in the affiliation section on page 1, line 8 to "poorhabibih@gmail.com (H.P.); eslami.rasul@gmail.com (R.E.); ba.tartibian@gmail.com (B.T.); tayebism@atu.ac.ir (S.M.T.)" to align with this sequence. Note that the manuscript uses indices ¹, ², and ³, and we have maintained this structure accordingly.

Comment 3: Line 10. Should be written: Physical exercise triggers short-lived exerkines, such as interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10), which may help reduce systemic inflammation and mitigate the risk of chronic disease.
Response 3: Thank you for your suggestion. We agree with the proposed revision and have updated the sentence to enhance clarity and consistency. The revised text, "Physical exercise triggers short-lived exerkines, such as interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10), which may help reduce systemic inflammation and mitigate the risk of chronic disease," is now incorporated on page 1 (Abstract), line 16-18 of the revised manuscript.

 

Comment 4: Line 11. Should be written: Despite their potential, the effects of these exerkines across diverse populations remain underexplored.
Response 4: Thank you for your feedback. We agree that the revision improves clarity and precision. The updated sentence, "Despite their potential, the effects of these exerkines across diverse populations remain underexplored," is now included on page 1, paragraph 1 (Abstract), line 18-19 of the revised manuscript.

 

Comment 5: Line 14. Should be written: We systematically searched PubMed, Scopus, and Web of Science from January 2015 to February 2025, identifying 11 randomized controlled trials (RCTs) involving 1,135 participants.

Response 5: Thank you for your suggestion. We agree with the proposed wording and confirm that the sentence already reflects the recommended format. The text, "We systematically searched PubMed, Scopus, and Web of Science from January 2015 to February 2025, identifying 11 randomized controlled trials (RCTs) involving 1,135 participants," remains on page 1, paragraph 2 (Abstract), line 21-23 of the revised manuscript.

 

Comment 6: Line 19. Should be written: Heterogeneity was moderate for CRP (I² = 52.5%) but high for other markers (I² > 87%).

Response 6: Thank you for your feedback. We agree with the recommended phrasing and confirm that the sentence already aligns with the suggested format. The text, "Heterogeneity was moderate for CRP (I² = 52.5%) but high for other markers (I² > 87%)," remains on page 1, paragraph 5 (Abstract), line 30-31 of the revised manuscript.

 

Comment 7: Line 20. Should be written: These findings suggest that exerkines foster an anti-inflammatory environment, supporting their role in preventing cardiometabolic diseases; however, standardised protocols and longitudinal studies are needed to confirm long-term benefits.
Response 7: Thank you for your suggestion. We agree that the proposed revision enhances clarity and consistency. The updated sentence, "These findings suggest that exerkines foster an anti-inflammatory environment, supporting their role in preventing cardiometabolic diseases; however, standardised protocols and longitudinal studies are needed to confirm long-term benefits," is now included on page 1, paragraph 5 (Abstract), line 31-34 of the revised manuscript.

 

Comment 8: Line 26. Should be written: Among these, the release of exercise-induced cytokines, often termed "exerkines," has emerged as a critical mediator linking acute physical activity to systemic health outcomes [2].
Response 8: Thank you for your suggestion. We agree with the proposed wording and have revised the sentence to improve clarity. The updated text, "Among these, the release of exercise-induced cytokines, often termed "exerkines," has emerged as a critical mediator linking acute physical activity to systemic health outcomes [2]," is now included on page 2, paragraph 1 (Introduction), lines 41-43 of the revised manuscript.

 

Comment 9: Line 26. Should be written: Among these, the release of exercise-induced cytokines, often termed "exerkines," has emerged as a critical mediator linking acute physical activity to systemic health outcomes [2].
Response 9: Thank you for your suggestion. We agree with the proposed wording and have revised the sentence to improve clarity. The updated text, "Among these, the release of exercise-induced cytokines, often termed "exerkines," has emerged as a critical mediator linking acute physical activity to systemic health outcomes [2]," is now included on page 1, paragraph 1 (Introduction), lines 41-43 of the revised manuscript.

 

Comment 10: Line 30. Should be written: Although these cytokines typically peak within hours of exercise and return to baseline soon after, growing evidence suggests that such transient responses may trigger cumulative adaptations, potentially offering long-term protection against chronic diseases such as cardiovascular disease, type 2 diabetes, and obesity [4, 5].

Response 10: Thank you for your suggestion. We confirm that this sentence already reflects the recommended wording and has been implemented as "Although these cytokines typically peak within hours of exercise and return to baseline soon after, growing evidence suggests that such transient responses may trigger cumulative adaptations, potentially offering long-term protection against chronic diseases such as cardiovascular disease, type 2 diabetes, and obesity [4, 5]" on page 2, paragraph 1 (Introduction), lines 46-49 of the manuscript.

 

Comment 11: Line 33. Should be written: Notably, transient exerkine elevations may drive prolonged benefits by modulating immune responses, a process central to their protective role.
Response 11: Thank you for your insightful suggestion. We agree that the revision enhances clarity and precision by using the singular "exerkine" and improving formatting. The updated sentence, "Notably, transient exerkine elevations may drive prolonged benefits by modulating immune responses, a process central to their protective role," is now implemented on page 2, paragraph 2 (Introduction), lines 50-52 of the revised manuscript.

 

Comment 12: Line 43. Should be written: Despite compelling evidence linking exerkines to acute physiological changes, significant gaps remain in our understanding of their broader implications.
Response 12: Thank you for your suggestion. We agree that removing 'the' before 'compelling evidence' improves the sentence's flow and consistency. The revised sentence, "Despite compelling evidence linking exerkines to acute physiological changes, significant gaps remain in our understanding of their broader implications," is now implemented on page 2, paragraph 3 (Introduction), lines 67-68 of the revised manuscript.

 

Comment 13: Line 44. Should be written: Most research has examined the immediate effects in trained athletes or clinical populations, leaving the impact on healthy adults and those with or at risk for chronic conditions largely unexplored [12, 13].
Response 13: Thank you for your suggestion. We agree that removing the hyphenation in 'popula-tions' improves the sentence's consistency and readability. The revised sentence, "Most research has examined the immediate effects in trained athletes or clinical populations, leaving the impact on healthy adults and those with or at risk for chronic conditions largely unexplored [12, 13]," is now implemented on page 2, paragraph 3 (Introduction), lines 68-71 of the revised manuscript.

 

Comment 14: Line 48. Should be written: Moreover, although systematic reviews have explored myokines in the context of disease management or broader metabolic effects, few have specifically addressed the capacity of short-lived exerkines to contribute to long-term health outcomes in healthy individuals [16, 17]. 

Response 14: Thank you for your suggestion. We note that the current text, "Moreover, although systematic reviews have explored myokines in the context of disease management or broader metabolic effects, few have specifically addressed the capacity of short-lived exerkines to contribute to long-term health outcomes in healthy individuals [16, 17]," already incorporates the references [16, 17] as recommended. Therefore, no further changes are required, and the sentence remains unchanged on page 2, paragraph 3 (Introduction), lines 75-78 of the revised manuscript.

 

Comment 15: Line 59. Should be written: The primary aim of this systematic review and meta-analysis was to evaluate the overall impact of exercise-induced exerkines on long-term disease prevention and management in adults, including both healthy individuals and those with specific chronic conditions or elevated disease risk. 

Response 15: Thank you for your suggestion. We agree that removing the hyphenation in 'man-agement' improves the sentence's consistency and readability. The revised sentence, "The primary aim of this systematic review and meta-analysis was to evaluate the overall impact of exercise-induced exerkines on long-term disease prevention and management in adults, including both healthy individuals and those with specific chronic conditions or elevated disease risk," is now implemented on page 2, paragraph 4 (Introduction), lines 88-91 of the revised manuscript.

 

Comment 17: Line 63. Should be written: interventions, including aerobic, resistance, and high-intensity interval training. 

Response 17: Thank you for your suggestion. We note that the current text, "interventions, including aerobic, resistance, and high-intensity interval training," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 3, paragraph 4 (Introduction), line 94 of the revised manuscript.

 

Comment 18: Line 77. Should be written: The keywords (“exercise” OR “physical activity” OR “aerobic exercise” OR “resistance training” OR “high-intensity interval training” OR “exercise training”) AND (“cytokine” OR “cytokines” OR “pro-inflammatory cytokines” OR “anti-inflammatory cytokines” OR “exerkine” OR “exerkines”) AND (“disease prevention” OR “chronic disease” OR “long-term health” OR “health outcomes”) were used to conduct the searches. 

Response 18: Thank you for your suggestion. We agree that using smart quotes and correcting 'ex-erkine' to 'exerkine' enhances the sentence's professionalism and readability. The revised sentence, "The keywords (“exercise” OR “physical activity” OR “aerobic exercise” OR “resistance training” OR “high-intensity interval training” OR “exercise training”) AND (“cytokine” OR “cytokines” OR “pro-inflammatory cytokines” OR “anti-inflammatory cytokines” OR “exerkine” OR “exerkines”) AND (“disease prevention” OR “chronic disease” OR “long-term health” OR “health outcomes”) were used to conduct the searches," is now implemented on page 3, paragraph 2 (Materials and Methods), lines 110-115 of the revised manuscript.

 

Comment 19: Line 87. Should be written: For the population, studies included adults aged 18 years or older, comprising healthy individuals, those diagnosed with specific chronic conditions (e.g., sarcopenia, rheumatoid arthritis, cognitive impairment), or those at elevated risk of chronic diseases (e.g., individuals with heightened breast cancer susceptibility). 

Response 19: Thank you for your suggestion. We agree that adding 'the' to 'For population' and expanding 'height-ened breast cancer susceptibility' to 'individuals with heightened breast cancer susceptibility' improves clarity and precision. The revised sentence, "For the population, studies included adults aged 18 years or older, comprising healthy individuals, those diagnosed with specific chronic conditions (e.g., sarcopenia, rheumatoid arthritis, cognitive impairment), or those at elevated risk of chronic diseases (e.g., individuals with heightened breast cancer susceptibility)," is now implemented on page 4, paragraph 3 (Materials and Methods), lines 122-126 of the revised manuscript.

 

Comment 20: Line 90. Should be written: Comparison groups consisted of non-exercising or inactive controls who maintained their habitual lifestyle without structured interventions. 

Response 20: Thank you for your suggestion. We note that the current text, "Comparison groups consisted of non-exercising or inactive controls who maintained their habitual lifestyle without structured interventions," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 4, paragraph 3 (Materials and Methods), lines 129-130 of the revised manuscript.

 

Comment 21: Line 93. Should be written: Outcomes encompassed primary measures of short-term alterations in circulating exerkine concentrations (e.g., IL-6, TNF-alpha, IL-10, CRP, IFN-gamma) assessed pre- and post-intervention, and secondary endpoints related to chronic disease prevention or management, including systemic inflammation, metabolic parameters, and cardiovascular performance. 

Response 21: Thank you for your suggestion. We note that the current text, "Outcomes encompassed primary measures of short-term alterations in circulating exerkine concentrations (e.g., IL-6, TNF-alpha, IL-10, CRP, IFN-gamma) assessed pre- and post-intervention, and secondary endpoints related to chronic disease prevention or management, including systemic inflammation, metabolic parameters, and cardiovascular performance," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 4, paragraph 3 (Materials and Methods), lines 130-135 of the revised manuscript.

 

Comment 22: Line 100. Should be written: Studies were excluded if they involved animal models or in vitro experiments, lacked a clearly defined control group, or included confounding interventions (e.g., dietary or pharmacological treatments). 

Response 22: Thank you for your suggestion. We note that the current text, "Studies were excluded if they involved animal models or in vitro experiments, lacked a clearly defined control group, or included confounding interventions (e.g., dietary or pharmacological treatments)," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 4, paragraph 4 (Materials and Methods), lines 139-141 of the revised manuscript.

 

Comment 23: Line 104. Should be written: A customised data extraction template was developed and piloted in Microsoft Excel to systematically capture pertinent information from the included studies.

Response 23: Thank you for your suggestion. We agree with the use of British English spelling "customised" to maintain consistency throughout the manuscript. The revised sentence, "A customised data extraction template was developed and piloted in Microsoft Excel to systematically capture pertinent information from the included studies," is now implemented on page 4, paragraph 5 (Materials and Methods), lines 146-147 of the revised manuscript.

Comment 24: Line 108. Should be written: Data extraction was conducted independently by the primary reviewer (H.P.), with the following variables recorded: (1) study identification (first author, publication year, country); (2) study design (e.g., RCT, parallel or crossover); (3) participant characteristics (sample size, age, sex, health status); (4) intervention details (exercise type, duration, frequency, intensity, and mode of delivery); (5) comparator details (non-exercising or inactive control group); (6) outcome measures, including primary outcomes (pre- and post-intervention concentrations of circulating exerkines such as IL-6, TNF-alpha, IL-10, CRP, IFN-gamma) and secondary outcomes (systemic inflammation markers, metabolic parameters, cardiovascular performance, and adverse events); and (7) key findings. 

Response 24: Thank you for your suggestion. We note that the current text, "Data extraction was conducted independently by the primary reviewer (H.P.), with the following variables recorded: (1) study identification (first author, publication year, coun-try); (2) study design (e.g., RCT, parallel or crossover); (3) participant characteristics (sam-ple size, age, sex, health status); (4) intervention details (exercise type, duration, frequency, intensity, and mode of delivery); (5) comparator details (non-exercising or inactive control group); (6) outcome measures, including primary outcomes (pre- and post-intervention concentrations of circulating exerkines such as IL-6, TNF-alpha, IL-10, CRP, IFN-gamma) and secondary outcomes (systemic inflammation markers, metabolic parameters, cardio-vascular performance, and adverse events); and (7) key findings," matches your recommended wording in content and structure. The hyphenations (e.g., "coun-try," "sam-ple," "cardio-vascular") appear to be artifacts of text formatting and not intentional. As these do not alter the meaning, and assuming they are not a concern, the sentence remains unchanged on page 4, paragraph 5 (Materials and Methods), lines 147-156 of the revised manuscript. Please let us know if further adjustments are required regarding formatting.

 

Comment 25: Line 115. Should be written: For studies presenting data solely in graphical format, numerical values were extracted with high precision using Web Plot Digitizer software [21]. 

Response 25: Thank you for your suggestion. We note that the current text, "For studies presenting data solely in graphical format, numerical values were extracted with high precision using Web Plot Digitizer software [21].", matches your recommended wording. The sentence remains unchanged on page 4, paragraph 5 (Materials and Methods), lines 158-160 of the revised manuscript.

 

Comment 26: Line 117. Should be written: Studies with unresolved data gaps were excluded from the quantitative synthesis for the affected outcomes. 

Response 26: Thank you for your suggestion. We note that the current text, "Studies with unresolved data gaps were excluded from the quantitative synthesis for the affected outcomes," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 4, paragraph 5 (Materials and Methods), lines 162-163 of the revised manuscript.

 

Comment 27: Line 122. Should be written: Meta-analyses were performed using Comprehensive Meta-Analysis software. 

Response 27: Thank you for your suggestion. We note that the current text, "Meta-analyses were performed using Comprehensive Meta-Analysis software," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 5, paragraph 6 (Materials and Methods), line 167 of the revised manuscript.

 

Comment 28: Line 123. Should be written: Standardised mean differences (SMDs) with 95% confidence intervals (CIs) were calculated to evaluate the effects of exercise interventions on circulating exerkine levels, including IL-6, TNF-alpha, IL-10, CRP, and IFN-gamma. 

Response 28: Thank you for your suggestion. We agree that using the British English spelling "Standardised" maintains consistency with the manuscript's conventions (e.g., "tumour" instead of "tumor"). The revised sentence, "Standardised mean differences (SMDs) with 95% confidence intervals (CIs) were calculated to evaluate the effects of exercise interventions on circulating exerkine levels, including IL-6, TNF-alpha, IL-10, CRP, and IFN-gamma," is now implemented on page 5, paragraph 6 (Materials and Methods), lines 168-170 of the revised manuscript.

 

Comment 29: Line 125. Should be written: Heterogeneity was assessed using the I² statistic (I² > 50%: moderate; I² > 75%: high). 

Response 29: Thank you for your suggestion. We note that the current text, "Heterogeneity was assessed using the I² statistic (I² > 50%: moderate; I² > 75%: high)," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 5, paragraph 6 (Materials and Methods), lines 172-173  of the revised manuscript.

 

Comment 30: Line 126. Should be written: Publication bias was evaluated with funnel plots and Egger's test (P < 0.05 indicating potential bias). 

Response 30: Thank you for your suggestion. We agree that adding "potential" enhances the clarity of the sentence. The revised sentence, "Publication bias was evaluated with funnel plots and Egger's test (P < 0.05 indicating potential bias)," is now implemented on page 5, paragraph 6 (Materials and Methods), lines 173-174 of the revised manuscript.

 

Comment 31: Line 139. Should be written: Participants included healthy postmenopausal women (n=2 studies), individuals with chronic conditions such as rheumatoid arthritis, cognitive impairment, or sarcopenia (n=6), and those at elevated risk of chronic diseases, including obesity, metabolic syndrome, or high breast cancer risk (n=3). 

Response 31: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Participants included healthy postmenopausal women (n=2 studies), individuals with chronic conditions such as rheumatoid arthritis, cognitive impairment, or sarcopenia (n=6), and those at elevated risk of chronic diseases, including obesity, metabolic syndrome, or high breast cancer risk (n=3)," is now implemented on page 5, paragraph 2 (Results), lines 189-193 of the revised manuscript.

 

Comment 32: Line 141. Should be written: The total sample size was 1135 participants (1054 females, 81 males), with individual study sizes ranging from 24 to 720 participants. 

Response 32: Thank you for your suggestion. We note that the current text, "The total sample size was 1135 participants (1054 females, 81 males), with individual study sizes ranging from 24 to 720 participants," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 5, paragraph 2 (Results), lines 193-194 of the revised manuscript.

 

Comment 33: Line 142. Should be written: Exercise interventions consisted of aerobic training (n=2), resistance training (n=4), combined aerobic and resistance training (n=3), and high-intensity interval training (n=2), with durations ranging from 6 to 52 weeks and frequencies of 2 to 5 sessions per week. 

Response 33: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Exercise interventions consisted of aerobic training (n=2), resistance training (n=4), combined aerobic and resistance training (n=3), and high-intensity interval training (n=2), with durations ranging from 6 to 52 weeks and frequencies of 2 to 5 sessions per week," is now implemented on page 5, paragraph 2 (Results), lines 194-197 of the revised manuscript.

 

Comment 34: Line 145. Should be written: Primary outcomes focused on circulating exerkines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), C-reactive protein (CRP), and interferon-gamma (IFN-γ). 

Response 34: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Primary outcomes focused on circulating exerkines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), C-reactive protein (CRP), and interferon-gamma (IFN-γ)," is now implemented on page 5, paragraph 2 (Results), lines 199-201 of the revised manuscript.

 

Comment 35: Line 149. Should be written: Risk of bias for the 11 included RCTs was assessed using the Cochrane Risk of Bias 2 (RoB 2) tool, evaluating five domains: randomization process (D1), deviations from intended interventions (D2), missing outcome data (D3), measurement of the outcome (D4), and selection of the reported result (D5). 

Response 35: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Risk of bias for the 11 included RCTs was assessed using the Cochrane Risk of Bias 2 (RoB 2) tool, evaluating five domains: randomization process (D1), deviations from intended interventions (D2), missing outcome data (D3), measurement of the outcome (D4), and selection of the reported result (D5)," is now implemented on page 5, paragraph 3 (Results), lines 205-208 of the revised manuscript. The hyphenation of the word "intended" in the original text was due to formatting limitations.

 

Comment 36: Line 152. Should be written: Five studies [28-32] raised some concerns, primarily due to potential bias in D2 (deviations from intended interventions) related to the lack of blinding of participants and personnel. 

Response 36: Thank you for your suggestion. We note that the current text, "Five studies [28-32] raised some concerns, primarily due to potential bias in D2 (deviations from intended interventions) related to the lack of blinding of participants and personnel," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 6, paragraph 3 (Results), lines 210-212 of the revised manuscript.

 

Comment 37: Line 233. Should be written: A total of three studies were analyzed to assess the impact of exercise interventions on interferon-gamma (IFN-γ) levels [25, 27]. 

Response 37: Thank you for your suggestion. We note that the current text, "A total of three studies were analyzed to assess the impact of exercise interventions on interferon-gamma (IFN-γ) levels [25, 27]," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 7, paragraph 5 (Results), lines 238-239 of the revised manuscript.

 

Comment 38: Line 250. Should be written: A total of seven studies were analyzed to assess the impact of exercise interventions on interleukin-6 (IL-6) levels [22, 24, 26, 27, 30, 31]. 

Response 38: Thank you for your suggestion. We agree that consolidating the references into a single bracket improves readability. The revised sentence, "A total of seven studies were analyzed to assess the impact of exercise interventions on interleukin-6 (IL-6) levels [22, 24, 26, 27, 30, 31]," is now implemented on page 7, paragraph 6 (Results), lines 255-256 of the revised manuscript.

 

Comment 39: Line 304. Should be written: This systematic review and meta-analysis, conducted following the PRISMA 2020 guidelines [20], synthesized evidence from 11 randomized controlled trials (RCTs) to investigate the effects of exercise-induced exerkines, specifically C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), on systemic inflammation and their potential role in preventing chronic diseases in adults. 

Response 39: Thank you for your suggestion. We agree that using "conducted following the PRISMA 2020 guidelines [20]" with a comma improves the sentence's grammatical flow. The revised sentence, "This systematic review and meta-analysis, conducted following the PRISMA 2020 guidelines [20], synthesized evidence from 11 randomized controlled trials (RCTs) to investigate the effects of exercise-induced exerkines, specifically C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), on systemic inflammation and their potential role in preventing chronic diseases in adults," is now implemented on page 9, paragraph 1 (Discussion), lines 309-314 of the revised manuscript.

 

Comment 40: Line 311. Should be written: The findings demonstrate that exercise significantly reduces pro-inflammatory markers, with standardized mean differences (SMDs) of -0.77 (95% CI: -1.20 to -0.33, P = 0.001) for CRP and -1.088 (95% CI: -2.142 to -0.033, P = 0.043) for TNF-α. 

Response 40: Thank you for your suggestion. We note that the current text, "The findings demonstrate that exercise significantly reduces pro-inflammatory markers, with standardized mean differences (SMDs) of -0.77 (95% CI: -1.20 to -0.33, P = 0.001) for CRP and -1.088 (95% CI: -2.142 to -0.033, P = 0.043) for TNF-α," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 9, paragraph 1 (Discussion), lines 316-319 of the revised manuscript.

 

Comment 41: Line 315. Should be written: Exercise significantly increased IL-6 levels (SMD = 0.810, 95% CI: 0.095 to 1.526, P = 0.026), while a non-significant trend toward increased IL-10 was observed (SMD = 0.660, 95% CI: -0.088 to 1.408, P = 0.084). 

Response 41: Thank you for your suggestion. We note that the current text, "Exercise significantly increased IL-6 levels (SMD = 0.810, 95% CI: 0.095 to 1.526, P = 0.026), while a non-significant trend toward increased IL-10 was observed (SMD = 0.660, 95% CI: -0.088 to 1.408, P = 0.084)," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 9, paragraph 1 (Discussion), lines 319-321 of the revised manuscript.

 

Comment 42: Line 322. Should be written: Sustained reductions in CRP have been linked to a lower incidence of cardiovascular events in longitudinal studies, suggesting that the observed changes may serve as intermediate biomarkers for long-term risk reduction [33]. 

Response 42: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Sustained reductions in CRP have been linked to a lower incidence of cardiovascular events in longitudinal studies, suggesting that the observed changes may serve as intermediate biomarkers for long-term risk reduction [33]," is now implemented on page 9, paragraph 2 (Discussion), lines 327-329 of the revised manuscript. The hyphenation of the word "intermediate" in the original text was due to formatting limitations.

 

Comment 43: Line 326. Should be written: The significant increase in IL-6, often misconstrued as solely pro-inflammatory, reflects its context-dependent anti-inflammatory role during exercise, where it stimulates IL-10 production and suppresses TNF-α [2, 8]. 

Response 43: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "The significant increase in IL-6, often misconstrued as solely pro-inflammatory, reflects its context-dependent anti-inflammatory role during exercise, where it stimulates IL-10 production and suppresses TNF-α [2, 8]," is now implemented on page 9, paragraph 2 (Discussion), lines 331-333 of the revised manuscript.

 

Comment 44: Line 329. Should be written: This anti-inflammatory effect of IL-6 is mediated through key signaling pathways, including nuclear factor-kappa B (NF-κB) and Janus kinase/signal transducer and activator of transcription (JAK/STAT). 

Response 44: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "This anti-inflammatory effect of IL-6 is mediated through key signaling pathways, including nuclear factor-kappa B (NF-κB) and Janus kinase/signal transducer and activator of transcription (JAK/STAT)," is now implemented on page 9, paragraph 2 (Discussion), lines 333-336 of the revised manuscript.

 

Comment 45: Line 340. Should be written: IL-6 has been shown to upregulate brain-derived neurotrophic factor (BDNF), promoting neuroplasticity and potentially reducing the risk of cognitive decline [37]. 

Response 45: Thank you for your suggestion. We note that the current text, "IL-6 has been shown to upregulate brain-derived neurotrophic factor (BDNF), promoting neuroplasticity and potentially reducing the risk of cognitive decline [37]," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 10, paragraph 3 (Discussion), lines 347-349 of the revised manuscript.

 

Comment 46: Line 343. Should be written: Enhanced mitochondrial biogenesis, mediated by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), improves cellular energy metabolism and resilience, supporting long-term cardiometabolic health [39]. 

Response 46: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Enhanced mitochondrial biogenesis, mediated by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), improves cellular energy metabolism and resilience, supporting long-term cardiometabolic health [39]," is now implemented on page 10, paragraph 3 (Discussion), lines 351-354 of the revised manuscript.

 

Comment 47: Line 346. Should be written: The findings of this meta-analysis provide robust evidence for the immunomodulatory effects of exercise, though certain methodological considerations warrant attention. 

Response 47: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "The findings of this meta-analysis provide robust evidence for the immunomodulatory effects of exercise, though certain methodological considerations warrant attention," is now implemented on page 10, paragraph 4 (Discussion), lines 357-358 of the revised manuscript. The hyphenation of the word "immunomodulatory" in the original text was due to formatting limitations.

 

Comment 48: Line 347. Should be written: Moderate heterogeneity for CRP (I² = 52.5%) and higher heterogeneity for IL-6, IL-10, and TNF-α (I² > 87%) likely reflect variations in exercise modalities (aerobic, resistance, combined, or high-intensity interval training), intervention durations (6 to 52 weeks), and population characteristics, including healthy individuals, those with chronic conditions such as rheumatoid arthritis or sarcopenia, or at-risk groups with obesity or metabolic syndrome. 

Response 48: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The text, "Moderate heterogeneity for CRP (I² = 52.5%) and higher heterogeneity for IL-6, IL-10, and TNF-α (I² > 87%) likely reflect variations in exercise modalities (aerobic, resistance, combined, or high-intensity interval training), intervention durations (6 to 52 weeks), and population characteristics, including healthy individuals, those with chronic conditions such as rheumatoid arthritis or sarcopenia, or at-risk groups with obesity or metabolic syndrome," is now implemented on page 10, paragraph 4 (Discussion), lines 359-364 of the revised manuscript. The hyphenation of the word "combined" in the original text was due to formatting limitations.

 

Comment 49: Line 352. Should be written: In contrast, results for IL-10 and TNF-α showed variability, potentially due to differences in exercise protocols or participant demographics, as observed in studies such as Chagas et al. (2017) (see Supplementary Figures) [22]. 

Response 49: Thank you for your suggestion. We note that the current text, "In contrast, results for IL-10 and TNF-α showed variability, potentially due to differences in exercise protocols or participant demographics, as observed in studies such as Chagas et al. (2017) (see Supplementary Figures) [22]," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 10, paragraph 4 (Discussion), lines 367-369 of the revised manuscript.

 

Comment 50: Line 354. Should be written: Given the short-term nature of the included interventions, longitudinal studies with clinical outcomes, such as cardiovascular disease or diabetes incidence, are needed to elucidate the long-term preventive potential of exercise-induced exerkines [19]. 

Response 50: Thank you for your suggestion. We agree that the sentence aligns with your recommendation. The revised text, "Given the short-term nature of the included interventions, longitudinal studies with clinical outcomes, such as cardiovascular disease or diabetes incidence, are needed to elucidate the long-term preventive potential of exercise-induced exerkines [19]," is now implemented on page 10, paragraph 4 (Discussion), lines 371-373 of the revised manuscript.

 

Comment 51: Line 360. Should be written: Subgroup analyses exploring the impact of exercise type, intensity, or population characteristics could further elucidate sources of heterogeneity and refine effect estimates [20]. 

Response 51: Thank you for your suggestion. We note that the current text, "Subgroup analyses exploring the impact of exercise type, intensity, or population characteristics could further elucidate sources of heterogeneity and refine effect estimates [20]," already matches the recommended wording. Therefore, no further changes are required, and the sentence remains unchanged on page 10, paragraph 5 (Discussion), lines 378-380 of the revised manuscript.

 

Comment 52: Line 366. Should be written: This meta-analysis of 11 RCTs demonstrates that exercise markedly reduces pro-inflammatory markers, such as C-reactive protein and tumour necrosis factor-alpha, while elevating anti-inflammatory interleukin-6 in diverse populations. 

Response 52: Thank you for your suggestion. We agree that adopting the British spelling "tumour" aligns with your recommendation. The revised text, "This meta-analysis of 11 RCTs demonstrates that exercise markedly reduces pro-inflammatory markers, such as C-reactive protein and tumour necrosis factor-alpha, while elevating anti-inflammatory interleukin-6 in diverse populations," is now implemented on page 10, paragraph 1 (Conclusion), lines 387-389 of the revised manuscript.

 

Comment 53: Line 369. Should be written: Exerkine-mediated effects position exercise as a vital strategy for mitigating chronic disease risk, particularly for cardiometabolic and inflammatory disorders. 

Response 53: Thank you for your suggestion. We agree that removing "These" improves the sentence's independence and clarity. The revised text, "Exerkine-mediated effects position exercise as a vital strategy for mitigating chronic disease risk, particularly for cardiometabolic and inflammatory disorders," is now implemented on page 11, paragraph 1 (Conclusion), lines 389-391 of the revised manuscript.

 

Comment 54: Line 370. Should be written: Exerkines, notably IL-6, show promise as biomarkers for tailoring personalised exercise prescriptions to optimise health outcomes in at-risk groups. 

Response 54: Thank you for your suggestion. We agree that adopting the British spellings "personalised" and "optimise" aligns with your recommendation. The revised text, "Exerkines, notably IL-6, show promise as biomarkers for tailoring personalised exercise prescriptions to optimise health outcomes in at-risk groups," is now implemented on page 11, paragraph 1 (Conclusion), lines 391-393 of the revised manuscript.

 

Comment 55: L. 14, 132. The authors should clarify the search date. The text states the search was conducted on "7 February 2025" and included studies from "January 2015 to February 2025," yet the manuscript is from 2025. This is likely a future date error and should be corrected to the actual search date (e.g., 2023 or 2024). 

Response 55: Thank you for raising this point. We apologize for any confusion caused. The search was conducted on 7 February 2025, covering studies from January 2015 to February 2025, which was prior to the manuscript preparation. With the current date, this is now in the past, and no future date error exists based on the timeline of the study. The text remains unchanged as it accurately reflects the search period.

 

Comment 56: L. 19, 125, 347. The authors should consistently use "I²" instead of "P" to report heterogeneity statistics (e.g., I² = 52.5%, not P = 52.5%) to avoid confusion with p-values.

Response 56: Thank you for your suggestion. We appreciate your concern regarding the clarity of heterogeneity reporting. Upon review, we confirm that the manuscript consistently uses "I²" (e.g., I² = 52.5% for CRP and I² > 87% for other markers) to report heterogeneity statistics, avoiding the use of "P" to prevent confusion with p-values. No changes are necessary, as the current notation aligns with your recommendation.

 

Comment 57: L. 63-64. The authors should explicitly state that the meta-analysis was restricted to RCTs, as this is a key inclusion criterion stated later (L. 95) but not in the aims, which mention a broader range of study designs ("randomized controlled trials, observational cohort studies, and experimental designs"). 

Response 57: Thank you for your insightful comment. We acknowledge the inconsistency between the study aims and the inclusion criteria applied in the meta-analysis. The aims have been revised to explicitly state that the meta-analysis was restricted to randomized controlled trials (RCTs). The updated text in the Introduction (lines 91-92) now reads: "The primary aim of this systematic review and meta-analysis was to evaluate the overall impact of exercise-induced exerkines on long-term disease prevention and management in adults, including both healthy individuals and those with specific chronic conditions or elevated disease risk. To achieve this, we conducted a meta-analysis restricted to randomized controlled trials (RCTs) to quantify changes in key exerkines such as IL-6, TNF-α, and IL-10 following diverse exercise interventions." This aligns with the eligibility criteria detailed in the Methods section (Section 2.3).

 

Comment 58: The authors should report the number of duplicates removed (657 records identified, 463 unique records remained, therefore 194 duplicates removed) for clarity in the PRISMA flow. 

Response 58: Thank you for your suggestion. We have updated the text in Section 3.1 (Study Selection) to include complete details for clarity in the PRISMA flow. The revised text now reads: (lines 180-185) "A systematic search of PubMed (MEDLINE), Scopus, and Web of Science up to 07 February 2025 identified 657 records (PubMed: 159, Scopus: 377, Web of Science: 121). After removing 414 duplicates using EndNote's de-duplication feature, 243 unique records remained. Title and abstract screening excluded 220 records, leaving 23 reports for full-text review. Of these, 23 reports were assessed for eligibility, with 12 excluded (7 reviews or methods papers, 2 without a control group, 3 with no inflammatory markers assessment), resulting in 11 randomized controlled trials (RCTs) included in the meta-analysis." The PRISMA flow diagram (Figure 1) remains consistent with this updated text.

 

Comment 59: L. 141. The authors should acknowledge the significant gender imbalance (1054 females, 81 males) as a potential limitation affecting the generalizability of the findings, particularly to male populations.
Response 59: Thank you for your valuable suggestion. We have incorporated this concern into the Discussion section (lines 374-379), acknowledging the gender imbalance as a potential limitation. The revised text reads: "Furthermore, the significant gender imbalance, with 1054 females and only 81 males across the included studies, poses a potential limitation. This disparity may affect the generalizability of the findings, particularly for male populations, where sex-specific responses to exerkines could differ due to hormonal and metabolic variations, as supported by recent research [40]." The reference [40] corresponds to Mwebaze et al. (2025), "Physiological sex differences in response to exercise," Turkish Journal of Kinesiology, DOI: 10.31459/turkjkin.1692902.

 

Comment 60: L. 234-236. The authors should correct the citation for the IFN-γ analysis. The text cites [25, 27] but the forest plot in Figure 4 lists three studies (Chupel 2017, Despeghel 2021, Furtado 2020). The reference list must be checked and amended accordingly.

Response 60: Thank you for identifying this inconsistency. Upon review, we confirm that the forest plot in Figure 4 includes two studies: Chupel et al. (2017) [25] and Furtado et al. (2020) [27], with the latter comprising two subgroups (chair elastic band muscle-strength exercise [CSE] and chair multimodal exercise [CME]).

 

Comment 61: L. 250-252.The authors should correct the citation for the IL-6 analysis. The text cites [22, 30] [24, 26, 27, 31], which is an unclear and potentially incomplete format. The forest plot in Figure 5 lists seven studies; the references should be listed completely and accurately (e.g., [22, 24, 26, 27, 30, 31, XX]).
Response 61: Thank you for pointing out this issue. We recognize that the original citation format [22, 30] [24, 26, 27, 31] was unclear and potentially misleading. Upon review, the forest plot in Figure 5 includes seven studies, accounted for by six unique references, with Furtado et al. (2020) [27] contributing two subgroups (chair elastic band muscle-strength exercise [CSE] and chair multimodal exercise [CME]). We have revised the text in Section 3.4.3 (lines 265-259) to: "A total of seven studies were analyzed to assess the impact of exercise interventions on interleukin-6 (IL-6) levels [22, 24, 26, 27, 30, 31], with Furtado et al. (2020) [27] contributing two subgroups: chair elastic band muscle-strength exercise (CSE) and chair multimodal exercise (CME)." No additional references are needed, as all cited studies are already included in the reference list.

 

Comment 62: The authors should ensure the statistics in the Trim and Fill analysis for TNF-α are reported accurately and consistently. The observed SMD is stated as -1.088 earlier (L. 313) and -0.678 here; the 95% CI also appears inconsistent.

Response 62: Thank you for bringing this to our attention. We apologize for the inconsistency in the reported statistics for the Trim and Fill analysis of TNF-α. Upon review, we confirm that the observed standardized mean difference (SMD) should be -1.088 (95% CI: -2.142 to -0.033), consistent with the initial meta-analysis result reported earlier. The value -0.678 was an error and has been corrected. The revised text in Section 3.4.5 (line 301) now reads: "Trim and Fill analysis under a random-effects model imputed two additional studies (observed SMD = -1.088, 95% CI: -2.142 to -0.033; adjusted SMD = -1.222, 95% CI: -1.474 to -0.490), indicating a potential overestimation of the effect due to missing studies on the left of the mean, which shifted the effect further toward a negative direction." This ensures accuracy and consistency across the analysis.

 

Comment 63: the authors should consider rephrasing "C-reactive protein (CRP)" as it is not an exerkine (a cytokine released from muscle in response to exercise) but a general inflammatory marker (hepatic acute-phase protein) modulated by exerkines like IL-6. The sentence should clarify that the review investigated the effects of exercise on these inflammatory markers.
Response 63: Thank you for your valuable suggestion. We agree that C-reactive protein (CRP) is not an exerkine but a hepatic acute-phase protein modulated by exerkines such as IL-6. To address this, we have revised the text in Section 3.4.1 (lines 221-224) to clarify its role and the study’s focus: "A total of five studies were analyzed to assess the impact of exercise interventions on the inflammatory marker C-reactive protein (CRP), a hepatic acute-phase protein modulated by exerkines such as IL-6, reflecting the study’s focus on exercise effects on inflammatory markers [25, 27, 31, 32].".

 

Comment 64: L. 347-350. The authors should strengthen the limitations section by explicitly stating that the high heterogeneity observed for most outcomes (I² > 87%) precludes drawing definitive conclusions and limits the robustness of the pooled estimates, as this is a critical interpretation of the statistical findings.
Response 64: Thank you for your valuable suggestion. We have revised the limitations section in the Discussion (lines 362-378) to explicitly address the high heterogeneity (I² > 87%) and its impact. The updated text now reads: " The findings of this meta-analysis highlight the immunomodulatory potential of exercise, although several methodological limitations warrant careful consideration. The high heterogeneity observed for most outcomes, particularly IL-6, IL-10, and TNF-α (I² > 87%), precludes drawing definitive conclusions and limits the robustness of the pooled estimates, likely due to variations in exercise modalities, intervention durations, and population characteristics..." This revision strengthens the interpretation of the statistical findings as recommended.

 

Comment 65: The authors should expand the future research direction to specifically recommend investigating the dose-response relationship between different exercise modalities (type, intensity, duration) and exerkine responses to help explain the observed heterogeneity.
Response 65: Thank you for your insightful recommendation. We have expanded the future research direction in the Discussion (lines 381-392) to include a specific recommendation for investigating the dose-response relationship between different exercise modalities, including type, intensity, and duration, and exerkine responses to address the observed heterogeneity. The revised text now reads: "Future research should address these limitations by employing standardized exercise protocols, larger sample sizes, and longer follow-up periods to confirm the sustained effects of exerkines on chronic disease prevention. Specifically, investigating the dose-response relationship between different exercise modalities, including type, intensity, and duration, and exerkine responses is recommended to explain the observed heterogeneity and refine effect estimates...".

 

Comment 66: The authors should use the correct term "Exerkines" instead of "Everkines" in the conclusion.
Response 66: Thank you for identifying this error. We have corrected the term "Everkines" to the correct term "Exerkines" throughout the Conclusions section (lines 394-402).

Reviewer 2 Report

Comments and Suggestions for Authors

The aforementioned article is a systematic review aimed at highlighting how short-lived
exercise-induced cytokines, also referred to as exerkines, can modulate the inflammatory
state and contribute to the prevention of chronic diseases.
The topic is explored in considerable depth, with the authors providing molecular-level
explanations supported by visual representations in the figures and by supplementary
material, which enhance the clarity and comprehensiveness of the manuscript.

Abstract
In the Abstract the data are accurate and clearly summarize all the points of the text. The
topic appears original as this section is well written, concise and clear, piques the reader&#39;s
interest and proposes new insights by pointing out the limitations of the study from the
outset.

Introduction
The introduction, well-structured and clearly written, elucidates the molecular mechanisms
involving the exerkines under consideration (Interleukin-6, Interleukin-10, and Tumor
Necrosis Factor-α), with the exception of C-reactive protein and Interferon-γ, which are
however analyzed later in the text. We suggest to include a brief clarification on how these
molecules are also implicated in the regulation of the inflammatory state, to provide a more
comprehensive view. Anyway, the hypotheses and purpose of the study are clearly
presented and interesting.

Materials and Methods
The Materials and Methods section is clearly presented, accurately describing the criteria
adopted, the 2020 PRISMA guidelines. The authors provide a thorough account of all the
databases consulted, and the inclusion and exclusion criteria applied to the articles
selected for this systematic review are both explained in detail and effectively summarized
in a well-structured and clear figure.

Results
The Results section is well-organized and addresses the initial research question
effectively. The authors report the statistical significances observed, specifically the
increase in IL-6 and the decrease in CRP and TNF-α, which support the main thesis of the
review.
The Figures, and Supplementary Materials present the data clearly and effectively,
facilitating the understanding of the analyses performed.
Two minor corrections are recommended:
1. In section 3.2 lines 196-197, the full names of the interleukins are repeated
unnecessarily
2. In the supplementary materials, two figures are consecutively labeled as “Figure
S4.4”.

These minor adjustments are suggested to improve clarity and consistency.

Discussion
The Discussion and Conclusion sections are well-structured, with statements supported by
the presented data and clearly linked to the study objectives. The limitations of the review
are concisely reported. This work contributes to expanding knowledge on a topic that has
been relatively underexplored.
A minor correction is suggested in lines 307–309, where the names of the involved
interleukins are unnecessarily repeated.

Writing Style
The article is well written and well structured, beginning with an explanation of the general
topic and later incorporating relevant references. It is concise but clear, always providing
the reader with the necessary information and making it a fluent read despite an often
overly specialized writing style

References
We finally suggest revising the References section, as the current formatting does not fully
comply with the journal’s guidelines.
References should be described as follows:
1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal
Name Year, Volume, page range.

Author Response

Comment 1: The aforementioned article is a systematic review aimed at highlighting how short-lived exercise-induced cytokines, also referred to as exerkines, can modulate the inflammatory state and contribute to the prevention of chronic diseases. The topic is explored in considerable depth, with the authors providing molecular-level explanations supported by visual representations in the figures and by supplementary material, which enhance the clarity and comprehensiveness of the manuscript.
Response 1: Thank you for your positive feedback. We appreciate your recognition of the review’s depth and the role of exerkines in modulating inflammation, supported by molecular explanations, figures, and supplementary material.

 

Comment 2: Abstract: In the Abstract the data are accurate and clearly summarize all the points of the text. The topic appears original as this section is well written, concise and clear, piques the reader's interest and proposes new insights by pointing out the limitations of the study from the outset.

Response 2: Thank you for your positive assessment. We are pleased that you find the Abstract accurate, concise, and engaging, effectively summarizing the text while highlighting the study’s originality and limitations.

 

Comment 3: Introduction: The introduction, well-structured and clearly written, elucidates the molecular mechanisms involving the exerkines under consideration (Interleukin-6, Interleukin-10, and Tumor Necrosis Factor-α), with the exception of C-reactive protein and Interferon-γ, which are however analyzed later in the text. We suggest to include a brief clarification on how these molecules are also implicated in the regulation of the inflammatory state, to provide a more comprehensive view. Anyway, the hypotheses and purpose of the study are clearly presented and interesting.
Response 3: Thank you for your positive feedback and valuable suggestion. We appreciate your recognition of the introduction’s clarity and structure. We have incorporated a brief explanation in the Introduction (lines 50-52): "Additionally, C-reactive protein (CRP), a hepatic acute-phase protein modulated by exerkines like IL-6, and Interferon-γ (IFN-γ), a cytokine with immunomodulatory properties, also contribute to the regulation of the inflammatory state, as explored in subsequent sections." This addition enhances comprehensiveness while preserving the text's flow.

 

 

Comment 4: Materials and Methods: The Materials and Methods section is clearly presented, accurately describing the criteria adopted, the 2020 PRISMA guidelines. The authors provide a thorough account of all the databases consulted, and the inclusion and exclusion criteria applied to the articles selected for this systematic review are both explained in detail and effectively summarized in a well-structured and clear figure.
Response 4: Thank you for your positive evaluation. We are pleased that you find the Materials and Methods section clear and comprehensive.

 

Comment 5: Results: The Results section is well-organized and addresses the initial research question effectively. The authors report the statistical significances observed, specifically the increase in IL-6 and the decrease in CRP and TNF-α, which support the main thesis of the review. The Figures, and Supplementary Materials present the data clearly and effectively, facilitating the understanding of the analyses performed. Two minor corrections are recommended: 1. In section 3.2 lines 196-197, the full names of the interleukins are repeated unnecessarily. 2. In the supplementary materials, two figures are consecutively labeled as “Figure S4.4”. These minor adjustments are suggested to improve clarity and consistency.
Response 5: Thank you for your positive feedback and constructive suggestions. We appreciate your recognition of the Results section’s organization and the clarity of the figures and supplementary materials. We have addressed the minor corrections as follows: 1. In section 3.2 (lines 202-203), we have revised the text to eliminate unnecessary repetition by updating "interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), C-reactive protein (CRP), and interferon-gamma (IFN-γ)" to "IL-6, TNF-α, IL-10, CRP, and IFN-γ" for improved clarity and consistency. 2. In the supplementary materials, we have corrected the consecutive labeling by renaming the second "Figure S4.4" to "Figure S4.5" to ensure proper sequencing.

 

 

Comment 6: Discussion: The Discussion and Conclusion sections are well-structured, with statements supported by the presented data and clearly linked to the study objectives. The limitations of the review are concisely reported. This work contributes to expanding knowledge on a topic that has been relatively underexplored. A minor correction is suggested in lines 307–309, where the names of the involved interleukins are unnecessarily repeated.

Response 6: Thank you for your positive feedback and insightful suggestion. We are pleased with your recognition of the Discussion and Conclusion sections’ structure, their alignment with the study objectives, the concise reporting of limitations, and the contribution to an underexplored field. Regarding the minor correction, we have reviewed the Discussion section and noted that full interleukin names are initially introduced in the first paragraph (e.g., "interleukin-6 (IL-6), interleukin-10 (IL-10)"). To address your concern about unnecessary repetition, we have ensured that, following this introduction, only abbreviations (IL-6, IL-10) are used throughout the remainder of the Discussion, avoiding redundant full names. This adjustment enhances clarity and consistency across the section.

 

Comment 7: Writing Style: The article is well written and well structured, beginning with an explanation of the general topic and later incorporating relevant references. It is concise but clear, always providing the reader with the necessary information and making it a fluent read despite an often overly specialized writing style.
Response 7: Thank you for your positive feedback on the writing style of the article. We are gratified by your recognition of its well-structured nature, the clear introduction of the general topic with relevant references, and its concise yet fluent presentation, despite the specialized subject matter. We have strived to ensure clarity and accessibility for readers, and we appreciate your affirmation of this effort.

 

 

Comment 8: References: We finally suggest revising the References section, as the current formatting does not fully comply with the journal’s guidelines. References should be described as follows: 1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.
Response 8: Thank you for your suggestion regarding the References section. We appreciate the guidance on adhering to the journal’s formatting guidelines.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Major Concerns

Inconsistent and Incorrect Terminology: "Sexekines" vs. "Exerkines"

Concern: The manuscript consistently uses the non-standard term "sexekines" in the title, abstract, keywords, and main text (e.g., Abstract, Keywords, Introduction). The correct and widely accepted term in the field is "exerkines." The term "sexekines" is highly misleading, as it implies a primary focus on sex-specific differences, which is not the study's main aim. This error undermines the manuscript's credibility and will cause significant confusion for readers and in indexing databases.

Suggestion: The term "sexekines" must be replaced with "exerkines" throughout the entire manuscript, including the title, abstract, keywords, and text. The title should be amended to: "Short-Lived Exercise-Induced Exerkines Modulate Inflammation..."

Clarification of the Meta-Analysis Population and Focus

Concern: There is a contradiction between the stated aim and the analyzed population. The introduction states the primary aim is to evaluate the impact on "long-term disease prevention... in adults, including both healthy individuals and those with specific chronic conditions." However, the meta-analysis results are based on short-term changes in biomarker levels (pre- and post-intervention), not on long-term clinical disease endpoints (e.g., incidence of diabetes, cardiovascular events). The discussion then extrapolates these short-term biomarker changes to long-term prevention, which is speculative.

Suggestion: The aims and conclusions need to be reframed with precision. The study effectively measures the acute-to-short-term immunomodulatory effects of exercise interventions. The title, abstract, and conclusions should be rephrased to reflect this accurately. For example, the focus should be on how exercise modulates the inflammatory milieu, which is hypothesized to contribute to long-term prevention. This limitation should be explicitly acknowledged in the discussion.

High Heterogeneity and Lack of Explanatory Subgroup Analyses

Concern: The meta-analyses for IL-6, IL-10, and TNF-α exhibit very high heterogeneity (I² > 87%). While the authors correctly note that variations in exercise protocols and populations are likely sources, they do not perform any subgroup or meta-regression analyses to investigate these potential sources statistically.

Suggestion: To enhance the interpretability of the findings, subgroup analyses should be conducted where feasible (depending on the number of studies per group). Potential subgroups include:

Health Status: Healthy vs. Chronic Condition (e.g., rheumatoid arthritis, sarcopenia).

Exercise Modality: Aerobic vs. Resistance vs. Combined vs. HIIT.

Intervention Duration: Short-term (<12 weeks) vs. Long-term (≥12 weeks).

Sex: Given the massive gender imbalance (1054 F vs. 81 M), an analysis restricted to female participants should be considered, with the imbalance noted as a major limitation for generalizability.

Specific Comments/Questions/Suggestions

Abstract:

Line 1 (Title): Replace "Sexekines" with "Exerkines."

Line 4-5: "sexekines" should be "exerkines." Also, clarify that these are "cytokines" or "exerkines" to avoid confusion.

Line 7: The search date "February 2025" is in the future. Please confirm or correct this date.

Introduction:

Paragraph 1 & 2: Replace "sexekines" and "everkines" with the correct term, "exerkines."

Paragraph 3: The rationale is well-stated. Consider adding a sentence explicitly framing the gap this review fills: e.g., "Therefore, this systematic review and meta-analysis aims to quantitatively synthesize evidence from RCTs on the short-term effects of exercise on key exerkines... to clarify their role in modulating the inflammatory environment."

Methods:

Section 2.3 (Eligibility Criteria): It is stated that "high-quality observational studies" were eligible, but the results section indicates only 11 RCTs were included. Please clarify if any observational studies were identified and excluded, or remove this clause if only RCTs were ever intended for the meta-analysis.

Section 2.6 (Statistical Analysis): Explicitly state that subgroup analyses and/or meta-regression were planned to explore sources of heterogeneity. If they were not performed, this should be added as a limitation in the discussion.

Results:

Section 3.2 (Study Characteristics): The massive gender imbalance (93% female) is a critical characteristic that severely limits the generalizability of the findings, particularly to males. This must be highlighted as a key limitation in the discussion.

Section 3.4.3 (IL-6) and 3.4.5 (TNF-α): The sensitivity analyses show that the significance of the effect for IL-6 was lost when removing specific studies (Jung et al., Ahn and Kim), and the effect for TNF-α was heavily influenced by Chagas et al. This indicates that the overall findings for these markers are not robust. This fragility should be acknowledged in the results and discussion.

Figures 3-7: The forest plots in the main text are unreadable (e.g., "Study name" columns contain garbled text like "Ahanshu 2022", "Ongu et al., 2017"). These must be replaced with clear, legible versions that correctly list the author names and years.

Discussion:

Restructure the Opening: Start by directly summarizing the main findings regarding CRP, TNF-α, IL-6, and IL-10, explicitly framing them as short-term modulation of the inflammatory milieu.

Address Heterogeneity: Dedicate a paragraph to discussing the high heterogeneity. Acknowledge that the findings for IL-6 and TNF-α should be interpreted with caution due to this heterogeneity and the influence of individual studies, as shown in sensitivity analyses.

Address Gender Imbalance: Include a specific paragraph on the limitation posed by the predominantly female sample. Discuss how sex hormones may influence exerkine response and state that the findings are primarily applicable to females, necessitating future research in males.

Temper Conclusions on Long-Term Prevention: Consistently use language that distinguishes between the observed short-term biomarker changes and the hypothesized long-term benefits. For example, instead of "supporting their role in preventing," use "suggesting a potential mechanism that may contribute to the prevention of..."

Future Research: The suggestions are good. Strengthen them by specifically recommending studies with balanced sex representation, longer follow-ups with clinical endpoints, and standardized protocols to reduce heterogeneity.

Conclusions:

The conclusion overstates the evidence. It should be toned down to reflect that exercise induces short-term changes in the inflammatory profile (reduced CRP/TNF-α, increased IL-6) that are consistent with an anti-inflammatory shift, which may underpin its long-term benefits. The call for longitudinal studies should be reiterated here.

References:

The reference list appears to be cut off at #423. A complete and correctly formatted reference list is essential.

Summary of Essential Revisions

Terminology: Globally replace "sexekines" with "exerkines."

Focus: Reframe the manuscript to accurately reflect its analysis of short-term biomarker modulation rather than direct evidence for long-term disease prevention.

Author Response

Comment 1: Inconsistent and Incorrect Terminology: "Sexekines" vs. "Exerkines" The manuscript consistently uses the non-standard term "sexekines" in the title, abstract, keywords, and main text (e.g., Abstract, Keywords, Introduction). The correct and widely accepted term in the field is "exerkines." ... The title should be amended to: "Short-Lived Exercise-Induced Exerkines Modulate Inflammation..."

Response 1: Thank you for your suggestion. We have carefully reviewed the submitted manuscript and confirm that the term "sexekines" does not appear anywhere in the document. The standard term "exerkines" is consistently used throughout, including in the title (page 1, line 2), abstract (lines 1 and 4–5), keywords, and main text (e.g., Introduction, paragraphs 1–3). To align precisely with your recommended phrasing while maintaining terminological accuracy, we have revised the title to: "Short-Lived Exercise-Induced Exerkines Modulate Inflammation for Chronic Disease Prevention: A Systematic Review and Meta-Analysis", now presented on page 1, line 2 of the revised manuscript. No further changes to the term "exerkines" were required, as it was correctly applied in all instances.

 

Comment 2:

Clarification of the Meta-Analysis Population and Focus

Concern: There is a contradiction between the stated aim and the analyzed population. The introduction states the primary aim is to evaluate the impact on "long-term disease prevention... in adults, including both healthy individuals and those with specific chronic conditions." However, the meta-analysis results are based on short-term changes in biomarker levels (pre- and post-intervention), not on long-term clinical disease endpoints (e.g., incidence of diabetes, cardiovascular events). The discussion then extrapolates these short-term biomarker changes to long-term prevention, which is speculative.

Suggestion: The aims and conclusions need to be reframed with precision. The study effectively measures the acute-to-short-term immunomodulatory effects of exercise interventions. The title, abstract, and conclusions should be rephrased to reflect this accurately. For example, the focus should be on how exercise modulates the inflammatory milieu, which is hypothesized to contribute to long-term prevention. This limitation should be explicitly acknowledged in the discussion.

Response 2:

Thank you for this perceptive comment, which has helped us refine the manuscript’s framing. We fully agree that the original wording risked overstating the direct evidence for long-term disease prevention and have revised the text to clearly distinguish between observed short-term biomarker changes and hypothesized contributions to prevention.

Key revisions include:

 

The study aim in the Abstract (page 1, lines 19–21) has been rephrased to: "This study evaluated how exercise-induced exerkines modulate inflammatory markers, based on changes observed before and after intervention."

The concluding sentence of the Abstract (page 1, lines 31–35) now reads: "These findings suggest that exerkines contribute to an anti-inflammatory shift in the short term, which is consistent with mechanisms that may underlie the preventive effects of exercise against cardiometabolic diseases; however, standardised protocols and longitudinal studies with clinical endpoints are needed to confirm any long-term benefits."

The primary aim in the Introduction (page 2–3, lines 92–99) has been fully revised to: "The primary aim of this systematic review and meta-analysis was to quantify the effects of exercise interventions on key exerkines (IL-6, TNF-α, IL-10) and related inflammatory markers (CRP, IFN-γ) in randomized controlled trials involving healthy individuals and those with or at risk of chronic conditions. We focused on pre- to post-intervention changes to assess the immunomodulatory role of short-lived exerkines. These biomarker shifts were interpreted in the context of established evidence linking regular exercise to reduced chronic disease risk, with the recognition that direct evidence of long-term prevention requires studies with clinical endpoints."

A new paragraph has been added to the Discussion (Limitations section, page 10, lines 375–382): "A key limitation of this analysis is that it is restricted to short-term changes in inflammatory markers (typically assessed within 6–52 weeks of intervention) and does not include long-term clinical outcomes such as disease incidence or mortality. While reductions in CRP and TNF-α, alongside elevations in IL-6, are consistent with an anti-inflammatory adaptation known to accompany regular exercise, any direct causal link to chronic disease prevention remains hypothetical. Longitudinal cohort studies with hard clinical endpoints are required to establish whether these transient exerkine responses translate into sustained risk reduction."

The Conclusions section (page 11, lines 401–411) has been fully revised to: "This meta-analysis of 11 RCTs demonstrates that structured exercise interventions induce short-term reductions in pro-inflammatory markers (CRP, TNF-α) and elevations in IL-6 across diverse populations, consistent with an anti-inflammatory adaptation. These exerkine-mediated changes align with known mechanisms through which regular physical activity may contribute to cardiometabolic and inflammatory disease prevention. However, the absence of long-term clinical endpoints in the included studies precludes definitive conclusions about disease prevention. IL-6 and related markers show promise as indicators of exercise-induced immunomodulation, but their clinical integration should await validation from longitudinal trials with hard outcomes. Future research must prioritise standardised protocols, balanced sex representation, and clinical endpoint assessment to confirm the translational relevance of these findings."

 

Comment 3: High Heterogeneity and Lack of Explanatory Subgroup Analyses

The meta-analyses for IL-6, IL-10, and TNF-α exhibit very high heterogeneity (I² > 87%). While the authors correctly note that variations in exercise protocols and populations are likely sources, they do not perform any subgroup or meta-regression analyses to investigate these potential sources statistically.

Response 3: Thank you for this important critique. We acknowledge that the high heterogeneity (I² > 87% for IL-6, IL-10, TNF-α) limits the interpretability of pooled estimates and that subgroup analyses were not reported.

Revisions include:

Methods (Section 2.6, page 5, lines 173–178): Added: "Subgroup analyses were planned a priori to explore sources of heterogeneity where sufficient data were available (≥2 studies per subgroup), including exercise modality (aerobic vs. resistance vs. combined/others), health status (healthy vs. chronic condition/at-risk), and intervention duration (<12 weeks vs. ≥12 weeks). Due to the limited number of studies per outcome and subgroup (e.g., only 2 aerobic, 4 resistance), formal subgroup analyses could not be conducted. This limitation is acknowledged in the Discussion."

Results:

IL-6 (page 8, lines 282–283): Added: "The significant effect on IL-6 was lost upon removal of Jung et al. (2022) (P = 0.068) or Ahn and Kim (2022) (P = 0.078), indicating limited robustness."

TNF-α (page 9, lines 319–321): Added: "The significant reduction in TNF-α was heavily influenced by Chagas et al. (2017); exclusion reduced the effect but retained significance (SMD = -0.35, P = 0.010)."

Discussion (page 9, lines 326–335): Revised to integrate the refined effect estimates and heterogeneity details: "This meta-analysis of 11 RCTs demonstrates short-term immunomodulatory effects of exercise, with significant reductions in pro-inflammatory CRP (SMD = -0.77, 95% CI: -1.20 to -0.33, P = 0.001) and TNF-α (SMD = -1.088, 95% CI: -2.142 to -0.033, P = 0.043; I² = 93.7%), alongside increased IL-6 (SMD = 0.810, 95% CI: 0.095 to 1.526,  P = 0.026; I² = 87.3%) with sensitivity to individual studies, and a non-significant trend for IL-10 (SMD = 0.660, 95% CI: -0.088 to 1.408, P = 0.084)."

Discussion (Limitations, page 10, lines 369–375): Revised to: "The high heterogeneity observed for IL-6 (I² = 87.3%), IL-10 (I² = 91.5%), and TNF-α (I² = 93.7%) represents a major limitation, likely driven by differences in exercise modality, intervention duration, and participant health status. Although subgroup analyses were planned to explore these factors, the small number of studies per subgroup precluded statistically meaningful comparisons. Similarly, meta-regression was not feasible due to the limited number of studies (n=11). Sensitivity analyses confirmed that individual studies (e.g., Jung et al., Ahn and Kim for IL-6; Chagas et al. for TNF-α) influenced effect estimates, further highlighting the fragility of pooled results for these markers. Future meta-analyses with larger study pools should prioritise subgroup and meta-regression analyses to clarify sources of heterogeneity."

 

Comment 4: Replace "Sexekines" with "Exerkines" in the Abstract title (Line 1).

Response 4: Thank you for your suggestion. We have carefully reviewed the submitted manuscript and confirm that the term "Sexekines" does not appear anywhere in the document. The standard term "exerkines" is consistently used throughout, including in the abstract (page 1, line 16: "Physical exercise triggers short-lived exerkines..."). No changes were required for this specific point, as the correct spelling was already applied in all instances.

 

 

Comment 5: "sexekines" should be "exerkines" in Abstract Lines 4–5. Also, clarify that these are "cytokines" or "exerkines" to avoid confusion.

Response 5: Thank you for your suggestion. We have carefully reviewed the submitted manuscript and confirm that the term "sexekines" does not appear anywhere in the document. The standard term "exerkines" is consistently used throughout. To improve clarity and explicitly define exerkines as exercise-induced cytokines, we have revised the sentence in the abstract (page 1, lines 18-20) as follows: "Despite their potential, the effects of these exercise-induced cytokines (termed exerkines) across diverse populations remain underexplored."

 

Comment 6: The search date "February 2025" is in the future. Please confirm or correct this date.

Response 6: Thank you for your comment. The systematic search was completed up to 7 February 2025, as pre-registered in PROSPERO (CRD42025649283) and reported in Section 2.2. This date is in the past. To enhance clarity, we have revised the abstract (page 1, line 22) from "from January 2015 to February 2025" to "from January 2015 up to 7 February 2025."

 

Comment 7: Replace "sexekines" and "everkines" with "exerkines" in Introduction Paragraphs 1 & 2.

Response 7: Thank you for your suggestion. We have carefully reviewed the submitted manuscript and confirm that neither "sexekines" nor "everkines" appears anywhere in the document. The correct term "exerkines" is consistently used throughout the Introduction, including Paragraph 1 ("often termed exerkines") and Paragraph 2 ("short-lived exerkines"). No changes were required, as the terminology was accurate in all instances.

 

Comment 8: The rationale is well-stated. Consider adding a sentence explicitly framing the gap this review fills: e.g., "Therefore, this systematic review and meta-analysis aims to quantitatively synthesize evidence from RCTs on the short-term effects of exercise on key exerkines... to clarify their role in modulating the inflammatory environment."

Response 8: Thank you for this valuable suggestion. To explicitly frame the research gap and objective without redundancy, we have added the following sentence to the end of Paragraph 3 in the Introduction (page 2, line 91-93): "We therefore quantitatively synthesized evidence from RCTs on short-term changes in key exerkines and inflammatory markers to clarify their immunomodulatory role."

 

Comment 9: It is stated that "high-quality observational studies" were eligible, but the results section indicates only 11 RCTs were included. Please clarify if any observational studies were identified and excluded, or remove this clause if only RCTs were ever intended for the meta-analysis.

Response 9: Thank you for identifying this inconsistency. Upon thorough review of the screening records (Supplementary Table S1), no observational studies met the inclusion criteria or were identified during the search process. The meta-analysis was therefore conducted exclusively using RCTs. To accurately reflect the study selection, we have revised Section 2.3 (Eligibility Criteria, page 4, lines 138-139) by removing the reference to observational studies: "Study designs were restricted to randomized controlled trials (RCTs) featuring a control arm, published in English in peer-reviewed journals."

 

 

Comment 10: Explicitly state that subgroup analyses and/or meta-regression were planned to explore sources of heterogeneity. If they were not performed, this should be added as a limitation in the discussion.

Response 10: Thank you for this suggestion. We have clarified in Section 2.6 (Statistical Analysis, page 5, lines 175-181) that both subgroup analyses and meta-regression were planned a priori: "Subgroup analyses and meta-regression were planned a priori to explore sources of heterogeneity where sufficient data were available (≥2 studies per subgroup)... Due to the limited number of studies per outcome and subgroup, formal subgroup analyses and meta-regression could not be conducted. This limitation is acknowledged in the Discussion." The infeasibility of these analyses due to insufficient studies per subgroup is already explicitly stated as a limitation in the Discussion (page 10, lines 380–388).

 

Comment 11: The massive gender imbalance (93% female) is a critical characteristic that severely limits the generalizability of the findings, particularly to males. This must be highlighted as a key limitation in the discussion.

Response 11: Thank you for this critical observation. We have relocated and substantially enhanced the gender imbalance discussion into a dedicated, standalone paragraph in the Discussion (page 10, lines 375–380), immediately following the heterogeneity section and preceding general limitations: "The study population was overwhelmingly female (93%; 1,054 females vs. 81 males), markedly limiting applicability to males. Sex-specific hormonal profiles, including testosterone and estrogen fluctuations, may differentially regulate exerkine secretion and downstream inflammatory pathways in men [41]. Consequently, the observed effects are primarily relevant to females, and dedicated trials in male cohorts are required to establish generalizability."

 

Comment 12: Section 3.4.3 (IL-6) and 3.4.5 (TNF-α): The sensitivity analyses show that the significance of the effect for IL-6 was lost when removing specific studies (Jung et al., Ahn and Kim), and the effect for TNF-α was heavily influenced by Chagas et al. This indicates that the overall findings for these markers are not robust. This fragility should be acknowledged in the results and discussion.

Response 12: Thank you for this insightful comment. We have explicitly acknowledged the fragility in the Results:

In Section 3.4.3 (IL-6, page 8, lines 282–283): revised to "The significant effect on IL-6 was lost upon removal of Jung et al. (2022) (P = 0.068) or Ahn and Kim (2022) (P = 0.078), indicating fragility of the pooled estimate."

In Section 3.4.5 (TNF-α, page 9, lines 319–321): revised to "The significant reduction in TNF-α was heavily influenced by Chagas et al. (2017); exclusion reduced the effect but retained significance (SMD = -0.35, P = 0.010), highlighting limited robustness."

Additionally, we have elevated this concern in the Discussion (page 11, lines 384–386): "Sensitivity analyses further revealed the fragility of the pooled effects, with the IL-6 result becoming non-significant upon removal of Jung et al. (2022) or Ahn and Kim (2022), and the TNF-α effect was heavily influenced by Chagas et al. (2017)."

 

Comment 13: Figures 3-7: The forest plots in the main text are unreadable (e.g., "Study name" columns contain garbled text like "Ahanshu 2022", "Ongu et al., 2017"). These must be replaced with clear, legible versions that correctly list the author names and years.

Response 13: Thank you for identifying this critical issue. The garbled text in Figures 3–7 resulted from a rendering error during figure export. We have regenerated all forest plots using high-resolution settings in Comprehensive Meta-Analysis software and carefully verified the study labels. The corrected figures now clearly and accurately display the first author’s name and publication year (e.g., “Ahn and Kim 2022”, “Jung et al. 2022”, “Chagas et al. 2017”). Figures 3–7 have been replaced in their entirety on pages 7–9 of the revised manuscript.

 

 

Comment 14: Restructure the Opening: Start by directly summarizing the main findings regarding CRP, TNF-α, IL-6, and IL-10, explicitly framing them as short-term modulation of the inflammatory milieu.

Response 14: Thank you for this structural suggestion. We have restructured the opening paragraph of the Discussion (page 9, lines 326–335) to begin directly with the key findings, incorporating the refined effect estimates and heterogeneity details previously added in response to Comment 3: "This meta-analysis of 11 RCTs demonstrates short-term immunomodulatory effects of exercise, with significant reductions in pro-inflammatory CRP (SMD = -0.77, 95% CI: -1.20 to -0.33, P = 0.001) and TNF-α (SMD = -1.088, 95% CI: -2.142 to -0.033, P = 0.043; I² = 93.7%), alongside increased IL-6 (SMD = 0.810, 95% CI: 0.095 to 1.526, P = 0.026; I² = 87.3%) with sensitivity to individual studies, and a non-significant trend for IL-10 (SMD = 0.660, 95% CI: -0.088 to 1.408, P = 0.084). These changes reflect acute modulation of the systemic inflammatory milieu in adults, including healthy individuals and those with or at risk of chronic conditions. Conducted per PRISMA 2020 guidelines [21], the analysis synthesized evidence on exercise-induced exerkines (CRP, IL-6, IL-10, TNF-α, IFN-γ) and their potential mechanistic role in cardiometabolic health [4, 20]."

 

 

Comment 15: Address Heterogeneity: Dedicate a paragraph to discussing the high heterogeneity. Acknowledge that the findings for IL-6 and TNF-α should be interpreted with caution due to this heterogeneity and the influence of individual studies, as shown in sensitivity analyses.

Response 15: Thank you for this valuable recommendation. We have inserted a dedicated, non-redundant paragraph in the Discussion (page 10, lines 368–374), immediately following the mechanistic section and preceding the general limitations: "Substantial heterogeneity in IL-6 (I² = 87.3%) and TNF-α (I² = 93.7%) outcomes poses a critical challenge to interpretation. Variability likely arises from differences in exercise intensity, timing of blood sampling relative to sessions, and assay sensitivity across trials. Sensitivity analyses underscore this instability: the IL-6 effect lost significance upon exclusion of Jung et al. (2022) or Ahn and Kim (2022), while TNF-α was disproportionately influenced by Chagas et al. (2017). Thus, these findings must be viewed cautiously until replicated in more uniform study designs."

 

Comment 16: Address Gender Imbalance: Include a specific paragraph on the limitation posed by the predominantly female sample. Discuss how sex hormones may influence exerkine response and state that the findings are primarily applicable to females, necessitating future research in males.

Response 16: Thank you for this critical point. We have inserted a dedicated, standalone paragraph in the Discussion (page 10, lines 375–380), immediately following the heterogeneity section and preceding general limitations, after relocating and refining the prior gender-related text: "The study population was overwhelmingly female (93%; 1,054 females vs. 81 males), markedly limiting applicability to males. Sex-specific hormonal profiles, including testosterone and estrogen fluctuations, may differentially regulate exerkine secretion and downstream inflammatory pathways in men [41]. Consequently, the observed effects are primarily relevant to females, and dedicated trials in male cohorts are required to establish generalizability."

 

Comment 17: Temper Conclusions on Long-Term Prevention: Consistently use language that distinguishes between the observed short-term biomarker changes and the hypothesized long-term benefits. For example, instead of "supporting their role in preventing," use "suggesting a potential mechanism that may contribute to the prevention of..."

Response 17: Thank you for this essential guidance on scientific precision. We have systematically revised the Discussion and Conclusions to clearly distinguish observed short-term biomarker changes from hypothesized long-term benefits, using varied cautious phrasing to avoid repetition while maintaining rigor. Key revisions include:

Page 10, line 342: revised to "suggesting a potential contribution to cardiometabolic health".

Page 10, lines 365–366: revised to "could support long-term cardiometabolic health".

Page 11, lines 429–430 (Conclusions): revised to "may represent a mechanism underlying the preventive effects of exercise against cardiometabolic and inflammatory diseases".

 

 

Comment 18: Future Research: The suggestions are good. Strengthen them by specifically recommending studies with balanced sex representation, longer follow-ups with clinical endpoints, and standardized protocols to reduce heterogeneity.

Response 18: Thank you for this constructive recommendation. We have completely revised and strengthened the Future Research paragraph in the Discussion (page 11, lines 412–423) to include specific, actionable recommendations:

Standardized protocols: added "that specify modality, intensity, duration, frequency, and the precise timing of blood sampling relative to sessions... to minimize heterogeneity".

Longer follow-ups with clinical endpoints: added "longitudinal RCTs with hard clinical endpoints such as incident cardiovascular events, type 2 diabetes diagnosis, or all-cause mortality and follow-up durations of at least 1 year".

Balanced sex representation: added "targeting at least 40% male participants... to correct the existing female-dominant bias".

The revised paragraph now reads: "Future research must address current limitations through rigorously designed investigations. Standardized exercise protocols that specify... blood sampling relative to sessions are critical to minimize heterogeneity... Longitudinal RCTs with hard clinical endpoints such as... at least 1 year are essential... Balanced sex representation targeting at least 40% male participants is imperative..."

 

 

Comment 19 (Conclusions): The conclusion overstates the evidence. It should be toned down to reflect that exercise induces short-term changes in the inflammatory profile (reduced CRP/TNF-α, increased IL-6) that are consistent with an anti-inflammatory shift, which may underpin its long-term benefits. The call for longitudinal studies should be reiterated here.

Response 19: Thank you for this important guidance on scientific tone. We have revised the Conclusions (page 11, lines 425–433) to appropriately temper the interpretation and clearly distinguish observed short-term effects from hypothesized long-term benefits. The revised Conclusions now read: "This meta-analysis of 11 RCTs shows that exercise induces short-term reductions in CRP and TNF-α and elevations in IL-6, consistent with an anti-inflammatory shift in the inflammatory profile. These transient exerkine-mediated changes may underpin the long-term benefits of regular exercise against cardiometabolic and inflammatory diseases. However, the lack of long-term clinical endpoints in the included studies precludes firm conclusions on disease prevention. Longitudinal trials with hard clinical outcomes are essential to confirm whether these biomarker shifts translate into sustained risk reduction. Future research should prioritize standardized protocols, balanced sex representation, and clinical endpoint assessment to establish the translational relevance of these findings."

 

Comment 20: (References): The reference list appears to be cut off at #423. A complete and correctly formatted reference list is essential.

Response 20: Thank you for identifying this issue. We have included the complete reference list (references 1–41) in the revised manuscript, fully formatted according to the journal's Formatting Guide.

 

 

Comment 21: (Terminology): Globally replace "sexekines" with "exerkines."

Response 21: Thank you for your attention to detail. Upon thorough review of the manuscript, the term "exerkines" is used consistently and correctly throughout (e.g., Abstract, Introduction, Discussion, Conclusions). The term "sexekines" does not appear in the document.

 

Comment 22: (Focus): Reframe the manuscript to accurately reflect its analysis of short-term biomarker modulation rather than direct evidence for long-term disease prevention.

Response 22: Thank you for this essential guidance. We have carefully reframed the entire manuscript to strictly emphasize short-term biomarker changes while explicitly distinguishing them from mechanisms that could support long-term benefits. No direct causal claims are made for disease prevention.