Review Reports - Explore the Optimal Treatment Regimen Across Combinations of Variate Protein Sources and Exercise Modalities and Its Associated Factors in Older Adults: A Network Meta-Analysis and Meta-Regression of Randomized Controlled Trials

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This meta-analysis summarizes the current knowledge about protein supplementation and exercise to fight sarcopenia. While the topic is relevant and the study is well designed, I have some comments to the authors:

line 57: The use of epimedic when referring to sarcopenia is imprecise. Strictly speaking, it is hard to apply this term to non-communicable diseases. I would change it for a more accurate word.
line 88: RT is not defined. It seems to refer to resistance training, but it should be especified.
line 110: NMA definition is misplaced in the main text. It should be moved from line 116 to line 110.
line 690: naval seems a typographical error
Table 1: acronyms for type of exercise should be included here (i.e., RET for resistance exercise training , etc)
- Under Comparison, A letter is overlapping a number
- Preferably, use International System units: 3-meter followed by (8-feet)
Protein supplementation (lines 310-321): Protein dosages have been compiled, but I think this information is very relevant for the comparisons. I strongly recommend to describe them in more detail, i.e., indicating the dosage for each protein source (it could be a supplmentary table)
Same for exercise: Crucial data, as duration, frequency, type of routine..., is lacking. This information must be included for meaningful comparisons.
Additional comparisons regarding effects in both sexes and age ranges will add on, taking into account participants' age spans over 30 years.

Author Response

Comments 1: line 57: The use of epidemic when referring to sarcopenia is imprecise. Strictly speaking, it is hard to apply this term to non-communicable diseases. I would change it for a more accurate word

Response 1:

Thank you for your comprehensive review and constructive comments. We removed the term “epidemic” and rephrase the statement as follows:

Lines 58-59

“Based on the rapidly growing rate of ageing population in worldwide, sarcopenia is becoming prevalent among older people, …”

Comments 2: line 88: RT is not defined. It seems to refer to resistance training, but it should be specified.

Response 2:

The statement is rephrased as follows:

Lines 89-91

“Especially, a high-protein diet or protein supplementation is believed to have surplus benefits in augmenting efficacy of exercise training for healthy older people [28-31], …”

Comments 3: line 110: NMA definition is misplaced in the main text. It should be moved from line 116 to line 110.

Response 3:

The NMA definition is revised accordingly (line 107, line 113).

Comments 4: line 690: naval seems a typographical error

Response 4:

The statement is rephrased as follows:

Lines 739-740

“In the present NMA, varieties of animal and plant proteins were pooling together and fully compared.”

Comments 5: Table 1: acronyms for type of exercise should be included here (i.e., RET for resistance exercise training , etc)

Response 5:

The acronyms for type of exercise are added in Table 1.

Comments 6: Under Comparison, A letter is overlapping a number

Preferably, use International System units: 3-meter followed by (8-feet)

Response 6:

Revised accordingly.

Comments 7: Protein supplementation (lines 310-321): Protein dosages have been compiled, but I think this information is very relevant for the comparisons. I strongly recommend to describe them in more detail, i.e., indicating the dosage for each protein source (it could be a supplmentary table)

Response 7:

The details regarding dosage for each protein source are presented in the supplementary Table S2. We added statements to clarify the information as follows:

Lines 344-348

“The detail information indicating dosage for each protein source is presented in Table S2. In summary, PS was prescribed by an average amount of 25 g (range 10–60 g) for whey protein, 22 g (range 5–43 g) for milk protein; 23 g (range 7–40 g) for soy protein, 19 g (range 9–40 g) for collagen; 20 g (range 10–40 g) for casein; 31 g (range 5–45 g) for meat, and 13 g (range 3–40 g) for rice protein.”

Comments 8: Same for exercise: Crucial data, as duration, frequency, type of routine..., is lacking. This information must be included for meaningful comparisons.

Response 8:

The details regarding dosage for each exercise protocol are presented in the supplementary Table S2. We added statements to clarify the information as follows:

Lines 355-365

“The detail information indicating dosage for each exercise is presented in Table S2. In summary, majority of the included trials (155 RCTs) employed RET by a moderate to high training load ≥ 70% one-repetition maximum (range 70–85%) or 6–9 of 10-point rating of perceived exertion. In addition, 43 RCTs employed AET by a moderate to high intensity ≥ 70% (70–90%) maximal heart rate or peak oxygen intake, as well as 6–8 of 10-point rating of perceived exertion. A total of 90 RCTs prescribed a low intensity for RET, AET, or MET. Regarding the training duration, in overall, 149 out of 235 RCTs conducted a short-term or medium-term training period ≤ 12 weeks (range 2–12 weeks) with a total of 6–84 training sessions (frequency, 2–7 sessions per week); the other 86 RCTs had an intervention period longer than 3 months (range 4–24 months) with a total of 25–1008 training sessions (frequency, 3–14 sessions per week).”

Comments 9: Additional comparisons regarding effects in both sexes and age ranges will add on, taking into account participants' age spans over 30 years.

Response 9:

We added sex and age ranges for meta-regression analysis. The details of analyzed results are presented in the supplementary Table S10. We added statements to indicate the information as follows:

Lines 260-264

“In addition, network meta-regression (NMR) models were used to identify any relevant moderators influencing heterogeneity across the studies. ... Potential moderators were identified on the basis of (1) participant characteristics, namely age range, BMI, sex (i.e., proportion of women in the sample), …”

In addition, the newly results are presented in section 3.9 of the Results part. The statements were rephrased as follows:

Lines 628-636 (section 3.9)

“In muscle mass, there was potential gender effect on relative treatment efficacy. Older age (ꞵ = –0.39; 95% CrI: –0.69, –0.09) as well as female sex (ꞵ = –0.33; 95% CrI: –0.60, –0.05) predicted minor effects on muscle mass gain; ...... In muscle strength, BMI (ꞵ = –0.26; 95% CrI: –0.52, –0.001) and age (ꞵ = –0.42; 95% CrI: –0.76, –0.06) may exert negative effects on relative treatment efficacy for handgrip and leg strength, respectively. …... For function outcome, younger age (ꞵ = 0.28; 95% CrI: 0.04, 0.52) and female sex (ꞵ = 0.61; 95% CrI: 0.23, 1.18) may have effects on increasing walking speed and chair rise, respectively.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a highly relevant topic in geriatric medicine and rehabilitation: the optimal combination of protein supplementation and exercise training for improving sarcopenia-related outcomes in older adults. The inclusion of a large number of randomized controlled trials and the application of a network meta-analysis (NMA) approach represent important strengths. However, several methodological and reporting aspects require clarification to ensure transparency, interpretability, and clinical applicability of the findings.

Abstract

The abstract is informative but quite dense. It would benefit from clearer structuring of the methods and results, particularly regarding the description of the included interventions.
Please specify the databases searched and the timeframe of the search.
Consider briefly clarifying the main outcomes assessed (e.g., lean mass, strength, functional performance measures).
While SUCRA rankings are reported, it should be emphasized that rankings should be interpreted alongside effect sizes and confidence intervals.

Introduction

The introduction appropriately frames sarcopenia as a major health issue in aging populations. However, the authors should more clearly identify the specific gap in the literature that justifies the present network meta-analysis.
It would be helpful to briefly discuss existing evidence comparing different protein sources (e.g., whey, casein, plant-based proteins) and their potential mechanistic differences in stimulating muscle protein synthesis.
Underline that numerous studies highlith the need to make easier the diagnosis or the assessment of saarcopenia risk factors, even if nutritions and supplementations are not yet considered

Methods

Please specify the databases searched, search terms, and the time period covered by the search strategy.
More detail is required on the risk-of-bias assessment of the included trials and how this was incorporated into the analysis.
It would also be useful to clarify how heterogeneity was evaluated and whether sensitivity analyses were conducted.

Results

The inclusion of 235 RCTs and over 20,000 participants is impressive; however, the characteristics of the included studies should be summarized clearly (e.g., participant age ranges, intervention duration, training intensity) in another table with result considerations.
The authors should clarify the definitions and measurement methods used for outcomes such as muscle mass and strength across studies (in another table).
While whey protein combined with resistance training ranks highest, the clinical relevance of the reported SMD values should be discussed.
Additional information on the network structure (e.g., network plots) and the distribution of interventions would improve transparency.

Discussion

The discussion appropriately highlights whey protein combined with resistance training as the most effective intervention. However, the authors should interpret these findings cautiously, given the heterogeneity across studies.
The potential mechanisms underlying the superiority of whey protein (e.g., leucine content, rapid digestion kinetics) could be discussed in more detail.
The authors should also consider practical implications, including adherence, cost, and feasibility in older populations.
The authors may also consider discussing the broader concept of targeted nutritional supplementation in rehabilitation, as optimizing specific nutrients may enhance functional recovery and physical performance in older adults. For example, previous studies have highlighted the potential benefits of supplements such as L-carnitine in improving physical performance and supporting rehabilitation outcomes. Integrating this perspective could help contextualize protein supplementation within a more comprehensive nutritional strategy for sarcopenia management (doi: 10.3390/jfmk6040093).

Conclusion

The conclusions are generally supported by the findings but should be moderated slightly, emphasizing that although whey protein combined with resistance training appears promising, individualized approaches and further high-quality RCTs are needed to confirm the optimal supplementation strategy for sarcopenia management.

Author Response

Comments 1: The manuscript addresses a highly relevant topic in geriatric medicine and rehabilitation: the optimal combination of protein supplementation and exercise training for improving sarcopenia-related outcomes in older adults. The inclusion of a large number of randomized controlled trials and the application of a network meta-analysis (NMA) approach represent important strengths. However, several methodological and reporting aspects require clarification to ensure transparency, interpretability, and clinical applicability of the findings.

Response 1:

Thank you for your comprehensive review and constructive comments. We revised our submitted manuscript point by point, according to the reviewer’s comments in following.

Abstract

Comments 2: The abstract is informative but quite dense. It would benefit from clearer structuring of the methods and results, particularly regarding the description of the included interventions.

Please specify the databases searched and the timeframe of the search.

Consider briefly clarifying the main outcomes assessed (e.g., lean mass, strength, functional performance measures).

While SUCRA rankings are reported, it should be emphasized that rankings should be interpreted alongside effect sizes and confidence intervals.

Response 2:

According to the reviewer’s comments, the statements in abstract are rephrased as follows:

Lines 27-46

“We comprehensively searched the electronic databases, namely PubMed, the Physiotherapy Evidence Database, EMBASE, the Cochrane Library, the China Knowledge Resource Integrated Database, CINAHL, and Google Scholar, from its inception until December 2025. We included randomized controlled trials (RCT) investigating the efficacy of PS by any protein sources in combination with three exercise types (resistance, aerobic, or multicomponent training) in untrained older adults. Main outcomes related to sarcopenia were assessed, including lean mass, handgrip as well as leg strength, and physical mobility measures. … The GRADE assessment framework was used to determine the certainty of evidence (CoE). We finally included 235 RCTs (20980 participants) for analyses. A total of 10 protein sources (whey, soy, casein, milk, and the others) were identified, corresponding to 24 monotherapy and combined regimens of PS and exercise. Among the treatment arms, whey plus resistance training was ranked as the most effective treatment for muscle mass (large SMD, 1.29; CoE, moderate) and leg strength (large SMD, 1.16; CoE, moderate); additionally, whey plus multicomponent exercise training achieved the most promising effects on such sarcopenia-related physical indicators as chair rise (large effect, SMD = 1.09; CoE: high), timed up and go (medium SMD, 0.70; CoE, high), and global mobility score (large SMD, 1.02; CoE, high).”

Introduction

Comments 3: The introduction appropriately frames sarcopenia as a major health issue in aging populations. However, the authors should more clearly identify the specific gap in the literature that justifies the present network meta-analysis.

It would be helpful to briefly discuss existing evidence comparing different protein sources (e.g., whey, casein, plant-based proteins) and their potential mechanistic differences in stimulating muscle protein synthesis.

Response 3:

According to the reviewer’s comments, the paragraphs in the introduction are rephrased as follows:

Lines 99-126

“The postprandial muscle protein synthetic response …. Due to that rate of digestibility and amount of leucine content are varied among different protein supplements, the muscle protein synthesis rates in response to the ingestion of such protein supplements may differ following exercise training. Previous research revealed that whey supplements stimulate higher postprandial muscle protein synthesis rates than soy [45] and casein [45, 46] do, especially in older individuals [47, 48]. In addition, the ingestion of soy after RET stimulates muscle protein synthesis to a greater extent when compared with casein [45], as well as the milk ingestion does while comparing with beef protein [49] and soy supplements [50], respectively. Reasonably, it is expected that such acutely superior effects of protein supplementation on postprandial muscle protein synthesis may lead to favorable long-term changes in training-induced skeletal muscle adaptation. However, it remains unclear whether the varied protein supplements obtain different long-term outcome of muscle health after exercise intervention. Identifying relative treatment efficacy among different protein supplements helps the clinical practitioners to efficiently make clinical decisions and set prompt intervention strategies.

A number of systematic reviews and network meta-analysis (NMA) studies have compared the effects among different protein sources of supplementation in young and older people [30, 48, 51-56], and results have generally indicated no significant difference in lean mass or strength gains while comparing whey with soy [51-53, 56] , milk [30, 56], and beef [54, 56], as well as soy versus other mixed protein sources [51, 56] during an exercise intervention. However, some other protein supplies were not yet fully compared with former protein supplements in an NMA study, such as collagen, rice, and oat as well as high-protein meal enriched of blended animal and plant protein in daily diets. Therefore, the purpose of this NMA study was to (1) compare the treatment efficacy across all combined regimen composed of different types of protein supply and training modality (i.e., RET, AET, and MET); (2) identify if there is any relevant moderator affecting the relative treatment efficacy; and (3) determine the optimal treatment regimen by ranking the probability of all cross-combined treatments for older adults.”

Comments 4: Underline that numerous studies highlight the need to make easier the diagnosis or the assessment of sarcopenia risk factors, even if nutritions and supplementations are not yet considered.

Response 4:

We made the statement in the introduction as follows:

Lines 69-74

“In addition, sarcopenia is commonly recognized as a multi-etiological disease …; and thereby, numerous authors have emphasized the consensus to make easier the diagnosis or the assessment of sarcopenia risk factors [15-17].”

Methods

Comments 5: Please specify the databases searched, search terms, and the time period covered by the search strategy.

Response 5:

We made the statement in the introduction as follows:

Lines 146-149

“We comprehensively searched electronic online databases—namely PubMed, the Physiotherapy Evidence Database (PEDro), EMBASE, the Cochrane Library, the China Knowledge Resource Integrated Database, CINAHL, and Google Scholar—from its inception until December 2025.”

Comments 6: More detail is required on the risk-of-bias assessment of the included trials and how this was incorporated into the analysis.

Response 6:

The statements regarding the risk-of-bias assessment and its incorporation for analyses were rephrased as follows:

Lines 225-232 (Section 2.6)

“Based on the rated scores across all six domains, an overall bias of each trial was determined to be low (all the seven items were rated as low-risk), moderate (any one high-risk item, combined with or without unclear-risk judged items), or high risk (two or more high-risk items) [60]. In addition, the individual bias score of each trial was incorporated into the analyses by computing the weighted bias score for each direct estimate in NMA [62], which was further used for bias account of a network estimate (described in the below section 2.8). Review Manager (RevMan) version 5.4.1 software was used to summarize the results of the risk-of-bias assessment.”

Lines 283-291 (Section 2.8)

“Additionally, we assessed the weighted contribution of direct comparisons to the NMA estimates for each outcome using the contributions matrix [62, 73, 74]. Firstly, the risk-of-bias assessment for each direct pairwise comparison was performed by the weighted computation of the individual bias score of each included trials [73]. In the following, the contributions matrix was used to calculate percentage contribution of all direct comparisons to the NMA estimates, indicating the contribution of direct evidence to the entire network [62, 73, 74]. Finally, the downgrading level for the study limitation was based on the amounts of contribution of each direct estimate and its corresponding risk-of-bias score to the NMA estimates [74].”

Comments 7: It would also be useful to clarify how heterogeneity was evaluated and whether sensitivity analyses were conducted.

Response 7:

The statements for evaluation of heterogeneity and inconsistency were presented as follows:

Lines 252-257 (Section 2.7)

“Network heterogeneity and inconsistency were assessed using the I2 and Q statistics alongside τ2 values to estimate variance across the studies. The consistency between direct and indirect comparisons was assessed using the node-splitting method [67]. Additionally, a full design-by-treatment interaction random-effects model was established to test global inconsistency across all treatment arms [68].”

Results

Comments 8: The inclusion of 235 RCTs and over 20,000 participants is impressive; however, the characteristics of the included studies should be summarized clearly (e.g., participant age ranges, intervention duration, training intensity) in another table with result considerations.

Response 8:

We added participant age ranges in Table 2.

The details regarding dosage for each exercise protocol are presented in the supplementary Table S2. We added statements to clarify the information as follows:

Lines 356-366

“The detail information indicating dosage for each exercise is presented in Table S2. In summary, majority of the included trials (155 RCTs) employed RET by a moderate to high training load ≥ 70% one-repetition maximum (range 70%–85%) or 6–9 of 10-point rating of perceived exertion. In addition, 43 RCTs employed AET by a moderate to high intensity ≥ 70% (70%–90%) maximal heart rate or peak oxygen intake, as well as 6–8 of 10-point rating of perceived exertion. A total of 90 RCTs prescribed a low intensity for RET, AET, or MET. Regarding the training duration, in overall, 149 out of 235 RCTs conducted a short-term or medium-term training period ≤ 12 weeks (range 2–12 weeks) with a total of 6–84 training sessions (frequency, 2–7 sessions per week); the other 86 RCTs had an intervention period longer than 3 months (range 4–24 months) with a total of 25–1008 training sessions (frequency, 3–14 sessions per week).”

Comments 9: The authors should clarify the definitions and measurement methods used for outcomes such as muscle mass and strength across studies (in another table).

Response 9:

We added a table (supplementary Table S3) to present all of the outcome measures across trials.

We also made statements to clarify the definitions and measurement methods used for outcomes as follows:

Lines 398-406 (section 3.6)

“All of the outcome measures across trials were presented in supplementary Table S3. The muscle mass was mostly measured by dual-energy X-ray absorptiometry (153 RCTs) or bioelectrical impedance analysis (33 RCTs), which were expressed as absolute whole-body lean mass, appendicular lean mass, or fat-free mass as well as its indices. In addition, muscle volume were expressed as cross-sectional area measured by magnetic resonance imaging (30 RCTs), muscle thickness measured by ultrasonography (19 RCTs), and muscle girth (14 RCTs) of arms or legs. When the trial reported multiple measures, priority for analysis was given to the following: lean body mass, fat-free mass, appendicular lean mass, cross-sectional area, muscle thickness, and girth.”

Lines 462-465 (section 3.7)

“Handgrip strength as well as leg strength was mostly measured by a handled dynamometer and expressed as maximal voluntary isometric contraction; other leg-strength measures included leg press (1, 5, 10 repetition maximum) and isokinetic knee extension (Table S3).”

Comments 10: While whey protein combined with resistance training ranks highest, the clinical relevance of the reported SMD values should be discussed. Additional information on the network structure (e.g., network plots) and the distribution of interventions would improve transparency.

Response 10:

We rephrased the statements to clarify the clinical relevance of the reported SMD values as follows:

Lines 432-435 (section 3.6.2)

“Pooling all treatment effects in NMA, WP+RET was ranked as the most effective (SUCRA = 0.93) by a large effect (SMD = 1.29) among all treatment arms for muscle mass gain—followed by regular Collagen+RET (SUCRA = 0.88; SMD = 1.23), and Meat+RET (SUCRA = 0.87; SMD = 1.16; Figure 4).”

Discussion

Comments 11: The discussion appropriately highlights whey protein combined with resistance training as the most effective intervention. However, the authors should interpret these findings cautiously, given the heterogeneity across studies.

Response 11: