The Effects of Non-Pharmacological Interventions in Fibromyalgia: A Systematic Review and Metanalysis of Predominants Outcomes

(1) Fibromyalgia (FM) is a chronic musculoskeletal condition with multiple symptoms primarily affecting women. An imbalance in cytokine levels has been observed, suggesting a chronic low-grade inflammation. The main aim of the meta-analysis was to examine the effect of multimodal rehabilitation on cytokine levels and other predominant variables in patients with FM. Furthermore, to examine which non-pharmacological tools have been used to investigate the effects that these can have on cytokines in FM patients. (2) Methods: Searches were conducted in PubMed, Scopus, Web of Science, Cochrane, and ScienceDirect databases. This systematic review and metanalysis followed the PRISMA statement protocol. The methodological quality of the studies was assessed using the PEDro scale, the risk of bias followed the Cochrane Manual 5.0.1, and the GRADE system was used for rating the certainty of evidence. (3) Results: Of 318 studies found, eight were finally selected, with a sample size of 320 women with a mean age of 57 ± 20. The proinflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α were the most studied. Resistance exercise, aquatic exercise, dynamic contractions, cycling, treadmill, and infrared therapy were the main non-pharmacological tools used. (4) Conclusions: The systematic review with meta-analysis found evidence of elevated cytokine levels in patients with FM, suggesting low chronic inflammation and a possible contribution to central sensitization and chronic pain. However, the effects of physiotherapeutic interventions on cytokine levels are variable, highlighting the importance of considering different factors and the need for further research.


Introduction
Fibromyalgia (FM) is characterized by chronic, widespread musculoskeletal pain with multiple tender points and generalized tenderness with muscle stiffness, joint stiffness, sleep disturbances, fatigue, mood, cognitive dysfunction, anxiety, depression, general tenderness, and inability to perform daily life activities.Regarding prevalence, it is estimated that 4% of the world's population is affected by FM, mainly in women aged 20-55 years [1].
The diagnosis is only clinical and consists of a complete assessment based on the 1990 American College of Rheumatology (ACR) criteria of three consecutive months of widespread pain and "tender points" of pain on palpation.In 2010, the ACR updated the criteria with two new parameters, and in 2016, the criteria were further revised to decrease the likelihood of misdiagnosis [2].
FM is a syndrome with a multifactorial etiology that develops depending on genetic predisposition, personal experiences, emotional and cognitive factors, and the individual's ability to cope with stress [3].

Search Strategies
Before starting the present systematic review, a search of different databases was carried out to verify the existence of recent reviews on the topic in question.Subsequently, several searches were performed using different combinations of the key terms, including "cytokines AND fibromyalgia" and "cytokines AND fibromyalgia AND physiotherapy".It used the search equation ("cytokines and Fibromyalgia and Physiotherapy") to focus the search on studies that used physiotherapy tools as an intervention and analyzed their possible effects on cytokines in FM patients.The same Medical Subject Headings (MeSH) terms were used to improve the specificity of the search.
A series of filters were established and included in each database to perform the current searches, which are detailed in Section 2.2 eligibility criteria.
The flow diagram below graphically shows the selection of the articles included in this systematic review and metanalysis.

Study Selection Process
In the present systematic review and metanalysis, the assessment of potentially relevant studies was performed based on their title and abstracts.The independent variables included in each of the selected studies were the number of patients, the number of withdrawals in each group, the clinical variables of the participants, the measurement tools evaluated, and the characteristics of the intervention applied.The primary dependent variable analyzed was the presence of proinflammatory and anti-inflammatory cytokines in plasma and muscle, regardless of the tool used to measure them.The secondary variables analyzed were the non-pharmacological techniques used.In total, eight studies were included in the systematic review and metanalysis.

Data Extraction Process
An exhaustive reading and evaluation of the eight studies finally selected were carried out, to which the PEDro scale was applied to assess their methodological quality, evaluating the design of the study, the source of obtaining the subjects, whether the study was randomized, whether there was concealment, whether there was blinding and what the outcome of the study was like.The PEDro scale of the synthesis results can be found in more detail in (Figure 1).was randomized, whether there was concealment, whether there was blinding and what the outcome of the study was like.The PEDro scale of the synthesis results can be found in more detail in (Figure 1).Also, the PRISMA 2020 checklist [12] was used to collect the most relevant data from each of the studies, author and year, type of study, sample characteristics, objectives, type of intervention, intervention time, number of sessions, frequency of sessions, session time, measures assessing the impact of FM, pain, fatigue, quality of life, depression, anxiety, physical capacity, blood tests to measure the different cytokines, results, conclusions, limitations of the study and the follow-up.The results of the data extraction will be presented in (Supplementary Materials Table S1).A reviewer working independently carried out both the study selection process and the data extraction process for each of the final articles.Subsequently, the results obtained were analyzed by two independent reviewers (IHB and EUD).In case of doubt or disagreement between the reviewers, they were jointly assessed until a consensus was reached.Also, the PRISMA 2020 checklist [12] was used to collect the most relevant data from each of the studies, author and year, type of study, sample characteristics, objectives, type of intervention, intervention time, number of sessions, frequency of sessions, session time, measures assessing the impact of FM, pain, fatigue, quality of life, depression, anxiety, physical capacity, blood tests to measure the different cytokines, results, conclusions, limitations of the study and the follow-up.The results of the data extraction will be presented in (Supplementary Materials Table S1).
A reviewer working independently carried out both the study selection process and the data extraction process for each of the final articles.Subsequently, the results obtained were analyzed by two independent reviewers (IHB and EUD).In case of doubt or disagreement between the reviewers, they were jointly assessed until a consensus was reached.

List of Data
The (Supplementary Materials Table S2) presents the table of the most studied cytokines, clearly identifying the most investigated and analyzed cytokines in the studies included in this review.On the other hand, (Supplementary Materials Table S3) contains a comparative table of cytokine levels between the control group of healthy subjects and the experimental group of subjects with FM, which will provide a more accurate and understandable picture of the differences between the two groups.

Risk of Bias Assessment of Individual Studies
Risk of bias is a tool developed by the Cochrane Collaboration to assess the methodology of scientific evidence.It is useful in systematic reviews for the individual analysis of included CTs and RCTs.In this sense, the present systematic review has followed the Cochrane Handbook 5.1.0[13] to assess the risk of bias.
The Cochrane Handbook 5.1.0presents six levels of bias: selection bias, conduct bias, detection bias, attrition bias, reporting bias, and other bias.Each level has one or more specific items in a Risk of Bias table, and each item includes a description of what happened in the study and an assessment where the assignment of "low risk", "high risk", or "unclear risk" of bias is included [13].The risk of bias assessment for each included study can be found in (Supplementary Materials Table S4) of this systematic review.

Synthesis Methods
The synthesis methods used in the present review are the eligibility criteria that were determined in Section 2.2 of material and methods and the analysis of methodological quality using the PEDro scale, which is based on the Delphi checklist developed by Verhagen [14].The checklist has a total of 11 items.The first item refers to external validity and is not considered for the final score; items 2-9 refer to internal validity, and items 10 and 11 indicate whether the statistical information provided by the authors allows for an adequate interpretation of the results.
Therefore, the maximum score is 10 points, and the minimum is 0. Only items that are answered affirmatively are scored.Studies with a score of 9-10 were of excellent methodological quality, 6-8 were of good quality, and 5 were of fair or acceptable quality.The PEDro scale can be found in more detail in Section 3.5 Results of the Synthesis.
Further to the synthesis measures, we assessed whether the studies included in the analysis met their objectives set at the start of the study.Of the eight studies included in this review, all of them met the objectives proposed at the outset.Regarding the homogeneity of the experimental and control groups of the studies, it was observed that in six of the eight articles [15][16][17][18][19][20], the groups were homogeneous, and the subjects were matched.However, in one of the studies [21], participants were not matched according to BMI, and the experimental group had higher BMI and blood pressure than the control group, and some participants had concomitant metabolic syndrome.In another study [22], the plasma analysis performed on the participants was not homogeneous, as the experimental group consisted of 75 subjects, while the control group had only 25 subjects.This information can be found in the descriptive tables in (Supplementary Materials Table S1).

Assessment of the Certainty of the Evidence
The GRADE system [23] was followed to measure the assessment of the certainty of the evidence, which defines the quality of evidence as the degree of confidence we have that the estimate of an effect is adequate to make a recommendation.In classifying the quality of evidence, the GRADE system establishes four categories: high, moderate, low, and very low.From the present systematic review, five of the eight studies have a high quality of evidence [17][18][19]21,22], and three studies have a moderate quality [15,16,20].These results can be seen in more detail in Section 3.6 Certainty of evidence.

Data Synthesis and Statistical Analysis
Statistical analysis of the meta-analysis was performed using the Review Manager software (RevMan 5.4; Cochrane Collaboration, Oxford, UK).A meta-analysis of the predominant variables using the same parameter and measurement scale was carried out using the random effects model, in which it was assumed that the effect of the treatments was not the same in all the studies included in the model.For this, the original values of each study (Mean and Standard Deviation or Median and interquartile range) were taken as reference.The effects of the experimental interventions against the comparison groups (controls, placebo, relaxation therapy, and healthy women without fibromyalgia) were presented as mean differences and their confidence intervals, taking a 95% CI as reference.The heterogeneity of the studies was evaluated using the I 2 statistic, where values greater than 35% were heterogeneous.The variance between studies was calculated using Tausquare (Tau2) [24].The significance level was set at 0.05 for statistically significant effects.
For the calculation of the effect size in cytokines, the predominant cytokine parameters at plasma levels (pg/mL) were selected and pooled from the original data of the different trials when a minimum N was obtained in two studies.Several trials reported median and interquartile ranges in their study.To combine the results of these studies, the mean and standard deviation of the sample were estimated using the method of Wan et al. [25].This method, less effective than the method proposed by Hozo et al. [26], was more suitable for such an estimate.In cases where studies reported only p value greater than 0.05 or mean difference (∆) in inter-and intragroup pre-and post-test value change, estimated values were taken from the original study data.Once all the necessary scores were obtained, the effect size of each parameter was calculated according to the method proposed by Lipsey and Wilson [27].When the hypothesis testing of the original trial employed nonparametric techniques or more than two means were compared, Eta squared, or Eta squared partial (η 2 ) was calculated directly or from Cohen's d using the method of Lenhard et al. [28].In some cases, the z-contrast statistic was calculated from the p-value offered in the trial.The cytokine meta-analysis model was random-effects based on the standardized mean difference and standard error of each study, also previously calculated [27].The size of the effect on cytokines was considered small around 0.01, about 0.06 was considered a medium effect, and greater than 0.14 a large effect, this being negligible when 0 was found in the CI [29].

Selection of Studies
During the initial stage of the search, 318 studies were identified from different databases.After removing duplicates, 302 studies remained.
To refine the selection, we applied date filters (2013-2023) and chose to select only clinical trials and randomized clinical trials, which were available in English or Spanish.After reviewing the titles and abstracts, 49 studies that did not fit the study topic were discarded, leaving ten studies for analysis.Of these, after a detailed reading, two studies were eliminated for inconclusive results, leaving a total of eight studies that met the inclusion criteria and were subjected to a qualitative analysis.
To carry out date and study type filters, we used database filters.To search for duplicates and perform the inclusion or exclusion of studies, we used an intelligent research collaboration platform called Rayyan, which optimizes efficiency in elaborating systematic reviews by facilitating the organization and classification of relevant studies to be considered.
All articles were clinical trials or randomized clinical trials, presenting baseline measures, and comparing them with post-intervention.Of the eight studies included in this systematic review, seven present an experimental group including subjects diagnosed with FM and a control group composed of healthy subjects [15][16][17][18][20][21][22]; within these seven studies, there are two that also compare two types of interventions [exercise and relaxation] in subjects with FM [21,22].A single study focuses on comparing the experimental group and a placebo group, while two studies compare two types of interventions (exercise and relaxation) in subjects with FM [19].More detailed information can be found in the descriptive table for each of the studies in (Supplementary Materials Table S1).

Risk of Bias in Individual Studies
A risk of bias assessment of the individual studies was performed, allowing a more accurate picture of the quality of the available evidence and the reliability of the results obtained.More detailed information on the risk of bias assessment of each of the studies included in this systematic review can be found in the tables in (Supplementary Materials Table S4).
The risk of bias assessment figures for each study included in this systematic review are shown below.Each figure will show the result of the risk of bias assessment for each domain assessed, allowing us to identify the strengths and weaknesses of each study.In this way, we will gain a more detailed understanding of the quality of the included studies and their impact on the overall results of the systematic review.
In the risk of bias graph (Figure 2), it can be seen that in the blinding of participants and staff, blinding of assessors, incomplete short-and long-term outcome data, and selective reporting is 100% low risk whereas, in the generation of the randomized sequence, the risk of bias is 100% low, while randomized sequence generation, allocation concealment and other sources of bias are 70% low risk, 62.5% low risk and 37.5% high risk.
between 35 and 66 years; the median age was around 50.All FM patients were diagnosed by ACR 1990 criteria.
All articles were clinical trials or randomized clinical trials, presenting baseline measures, and comparing them with post-intervention.Of the eight studies included in this systematic review, seven present an experimental group including subjects diagnosed with FM and a control group composed of healthy subjects [15][16][17][18][20][21][22]; within these seven studies, there are two that also compare two types of interventions [exercise and relaxation] in subjects with FM [21,22].A single study focuses on comparing the experimental group and a placebo group, while two studies compare two types of interventions (exercise and relaxation) in subjects with FM [19].More detailed information can be found in the descriptive table for each of the studies in (Supplementary Materials Table S1).

Risk of Bias in Individual Studies
A risk of bias assessment of the individual studies was performed, allowing a more accurate picture of the quality of the available evidence and the reliability of the results obtained.More detailed information on the risk of bias assessment of each of the studies included in this systematic review can be found in the tables in (Supplementary Materials Table S4).
The risk of bias assessment figures for each study included in this systematic review are shown below.Each figure will show the result of the risk of bias assessment for each domain assessed, allowing us to identify the strengths and weaknesses of each study.In this way, we will gain a more detailed understanding of the quality of the included studies and their impact on the overall results of the systematic review.
In the risk of bias graph (Figure 2), it can be seen that in the blinding of participants and staff, blinding of assessors, incomplete short-and long-term outcome data, and selective reporting is 100% low risk whereas, in the generation of the randomized sequence, the risk of bias is 100% low, while randomized sequence generation, allocation concealment and other sources of bias are 70% low risk, 62.5% low risk and 37.5% high risk.Furthermore, in the risk of bias summary (Figure 3), it can be seen which author and item has a low risk, unclear risk, and high risk.In the present review, clearly, those with the highest risk of bias are the non-randomized clinical trial studies [15,16,20], for the 0% 10% 20% 30% 40% 50% 60% 70% 80% 90%100% Furthermore, in the risk of bias summary (Figure 3), it can be seen which author and item has a low risk, unclear risk, and high risk.In the present review, clearly, those with the highest risk of bias are the non-randomized clinical trial studies [15,16,20], for the items of random sequence generation and allocation concealment, compared to the randomized clinical trial studies [17][18][19]21,22], which have in this case a low risk.Another item where a high risk of bias was found in three of the articles was in other sources of bias.In two of the studies [18,20], it was by allowing participants to continue taking the medication they were taking and controls to take medication during the intervention which could condition the results; in another study [21] it was by not matching subjects in the experimental group with subjects in the control group.

Results of the Synthesis
The articles included in the review were assessed using the PEDro methodological quality scale, shown below in Table 1.The final score obtained ranged from 5 to 10. Two studies were classified as being of excellent quality, three of good quality, and three of fair or fair quality.The studies achieved a mean value of 7.12 ± 2.88.

Certainty of Evidence
The GRADE system [23] has been used to classify the studies included in this review to determine the certainty of the evidence, five of the eight studies have a high quality of evidence [17][18][19]21,22], and three studies a moderate quality [15,16,20], and can be visualized more clearly in the following Table 2.

Metanalysis Results
Figure 4 shows the effects of the treatments versus the comparison group on the outcome measures.Ernberg et al. [20] showed a reduction of 7.1 points in the FIQ instrument.The treatments were effective in combination with the study by Slam et al. [19] [IV = −7.84(−13.09 to −2.60); z = 2.93; p = 0.003].Figure 5 shows the effects of the treatment compared to the comparison group on the algometry (PPT) in the left and right limbs.The study by Jablochkova et al. [21] found no statistically significant differences in the Algometer measurement in favor of the resistance program versus relaxation therapy (z = 0.19; p = 0.85), not observing intragroup effects on this variable in the original study.No heterogeneity was observed in either of the two analyses (χ 2 (1) = 0.89; I 2 = 0% and (χ 2 (1) = 0.86; I 2 = 0% respectively).1: elegibility criteria were specified; 2: subjects were ramdomly allocated to groups; 3: allocation was concealed; 4: the groups were similar at baseline regarding the most important prognostic indicators; 5: blinding of all subjects; 6: blinding of all therapist who administered the therapy; 7: blinding of all assessors who measured at least one key outcome; 8: >85% outcomes of the subjets initially allocated to groups; 9: data for at least one key outcome by "intention to treat"; 10: between-group statistical comparisons; 11: point measures and measures of variability; N/A: not available.

Certainty of Evidence
The GRADE system [23] has been used to classify the studies included in this review to determine the certainty of the evidence, five of the eight studies have a high quality of evidence [17][18][19]21,22], and three studies a moderate quality [15,16,20], and can be visualized more clearly in the following Table 2.  [20] Ramdomized clinical trial High Jablochkova et al., 2019 [21] Ramdomized clinical trial High Salm et al., 2019 [18] Ramdomized clinical trial High Torgrimson-Ojerio et al., 2014 [22] Clinical trial Moderate

Metanalysis Results
Figure 4 shows the effects of the treatments versus the comparison group on the outcome measures.Ernberg et al. [20] showed a reduction of 7.1 points in the FIQ instrument.The treatments were effective in combination with the study by Slam et al. [19] [IV = −7.84(−13.09 to −2.60); z = 2.93; p = 0.003].Figure 5 shows the effects of the treatment compared to the comparison group on the algometry (PPT) in the left and right limbs.The study by Jablochkova et al. [21] found no statistically significant differences in the Algometer measurement in favor of the resistance program versus relaxation therapy (z = 0.19; p = 0.85), not observing intragroup effects on this variable in the original study.No heterogeneity was observed in either of the two analyses (χ 2 (1) = 0.89; I 2 = 0% and (χ 2 (1) = 0.86; I 2 = 0% respectively).Likewise, a change of 2.5 points was found in the mental component of the SF-36 scale in favor of the group that underwent 15 weeks of progressive resistance exercise and in the overall combination in favor of treatment in the SF-36, the pain, and the variable Static strength knee extension force (Figure 6).No heterogeneity was observed in either of the FIQ, PPT, SF-36, and HADS analyses (I 2 = 0%), however, heterogeneity was observed from χ 2 (2)= 196.33; p < 0.01; I 2 = 99% for the pain variable; (χ 2 (2)= 196.33; p < 0.01; I 2 = 99%; for elbow flexion force and χ 2 (2) = 4.88; p = 0.09; I 2 = 59% for the outcome knee extension force.Figure 6 shows the effects of treatment versus comparison group on the QoL (SF-36-PSC, SF-36-MSC and combined effects).Ernberg et al. [20] found a significant increase Likewise, a change of 2.5 points was found in the mental component of the SF-36 scale in favor of the group that underwent 15 weeks of progressive resistance exercise and in the overall combination in favor of treatment in the SF-36, the pain, and the variable Static strength knee extension force (Figure 6).No heterogeneity was observed in either of the FIQ, PPT, SF-36, and HADS analyses (I 2 = 0%), however, heterogeneity was observed from χ 2 (2) = 196.33;p < 0.01; I 2 = 99% for the pain variable; χ 2 (2) = 196.33;p < 0.01; I 2 = 99%; for elbow flexion force and χ 2 (2) = 4.88; p = 0.09; I 2 = 59% for the outcome knee extension force.Figure 6 shows the effects of treatment versus comparison group on the QoL (SF-36-PSC, SF-36-MSC and combined effects).Ernberg et al. [20] found a significant increase in the mental component of the quality of life of these patients after treatment (IV = −6.90(−11.52 to −2.28)).The treatments were effective in combination with the study by Jablochkova et al. [21] (IV = −6.67 (−10.65 to −2.68); z = 3.28; p = 0.001).No statistically significant changes were found in the combination of values from the pre to post-test in quality of life in its physical dimension in any of the studies examined (p > 0.05) (Figure 7).Likewise, a change of 2.5 points was found in the mental component of the SF-36 scale in favor of the group that underwent 15 weeks of progressive resistance exercise and in the overall combination in favor of treatment in the SF-36, the pain, and the variable Static strength knee extension force (Figure 6).No heterogeneity was observed in either of the FIQ, PPT, SF-36, and HADS analyses (I 2 = 0%), however, heterogeneity was observed from χ 2 (2)= 196.33; p < 0.01; I 2 = 99% for the pain variable; (χ 2 (2)= 196.33; p < 0.01; I 2 = 99%; for elbow flexion force and χ 2 (2) = 4.88; p = 0.09; I 2 = 59% for the outcome knee extension force.Figure 6 shows the effects of treatment versus comparison group on the QoL (SF-36-PSC, SF-36-MSC and combined effects).Ernberg et al. [20] found a significant increase in the mental component of the quality of life of these patients after treatment (IV = −6.90(−11.52 to −2.28).The treatments were effective in combination with the study by Jablochkova et al. [21] (IV = −6.67 (−10.65 to −2.68); z = 3.28; p = 0.001).No statistically significant changes were found in the combination of values from the pre to post-test in quality of life in its physical dimension in any of the studies examined (p > 0.05) (Figure 7).The studies by Ernberg et al. [17] and Jablochkova et al. [21] found a significant reduction in pain in favor of the experimental group (IV = −20.00(−22.64 to −17.36) and IV = 16.80 (−26.88 to −6.72); p < 0.05, respectively.However, no statistically significant differences were found in the overall analysis of both studies (Figure 8).The studies by Ernberg et al. [17] and Jablochkova et al. [21] found a significant reduction in pain in favor of the experimental group IV = −20.00(−22.64 to −17.36) and IV = 16.80 (−26.88 to −6.72); p < 0.05, respectively.However, no statistically significant differences were found in the overall analysis of both studies (Figure 8).The studies by Ernberg et al. [17] and Jablochkova et al. [21] found a significant reduction in pain in favor of the experimental group (IV = −20.00(−22.64 to −17.36) and IV = 16.80 (−26.88 to −6.72); p < 0.05, respectively.However, no statistically significant differences were found in the overall analysis of both studies (Figure 8).The studies by Ernberg et al. [17] and Jablochkova et al. [21] found a significant reduction in pain in favor of the experimental group (IV = −20.00(−22.64 to −17.36) and IV = 16.80 (−26.88 to −6.72); p < 0.05, respectively.However, no statistically significant differences were found in the overall analysis of both studies (Figure 8).Analysis of treatment effects was not significant in our IL-8 analyses.In the second study by Ernberg et al. [20], no intergroup effects were observed (Exercise treatment vs. Relaxaxion group) because 0 was found in IQ (p > 0.05), although there was a trend in favor of treatment in the original study (Figure 11).Analysis of treatment effects was not significant in our IL-8 analyses.In the second study by Ernberg et al. [20], no intergroup effects were observed (Exercise treatment vs. Relaxaxion group) because 0 was found in IQ (p > 0.05), although there was a trend in favor of treatment in the original study (Figure 11).
No statistically significant differences were observed in the analysis of the effect of the studies on cortisol.Bote's trial [16] found a difference of 0.38, but it was not significant (p > 0.05), as was the TNF variable (Figures 12 and 13).The studies that examined the IL-1β variable found negligible effects in the trials included in the overall analysis conducted (p > 0.05) (Figure 14).Analysis of treatment effects was not significant in our IL-8 analyses.In the second study by Ernberg et al. [20], no intergroup effects were observed (Exercise treatment vs. Relaxaxion group) because 0 was found in IQ (p > 0.05), although there was a trend in favor of treatment in the original study (Figure 11).No statistically significant differences were observed in the analysis of the effect of the studies on cortisol.Bote's trial [16] found a difference of 0.38, but it was not significant (p > 0.05), as was the TNF variable (Figures 12 and 13).The studies that examined the IL-1β variable found negligible effects in the trials included in the overall analysis conducted (p > 0.05) (Figure 14).favor of treatment in the original study (Figure 11).No statistically significant differences were observed in the analysis of the effect of the studies on cortisol.Bote's trial [16] found a difference of 0.38, but it was not significant (p > 0.05), as was the TNF variable (Figures 12 and 13).The studies that examined the IL-1β variable found negligible effects in the trials included in the overall analysis conducted (p > 0.05) (Figure 14).However, a statistically significant effect size was found in the study by Ernberg et al. [20] in favor of relaxation treatment on IL-6 (SMD = 0.50 (0.08 to 0.92; p < 0.05) (Figure 15).However, a statistically significant effect size was found in the study by Ernberg et al. [20] in favor of relaxation treatment on IL-6 (SMD = 0.50 (0.08 to 0.92; p < 0.05) (Figure 15).However, a statistically significant effect size was found in the study by Ernberg et al. [20] in favor of relaxation treatment on IL-6 (SMD = 0.50 (0.08 to 0.92; p < 0.05) (Figure 15).[17,19].
However, a statistically significant effect size was found in the study by Ernberg et al. [20] in favor of relaxation treatment on IL-6 (SMD = 0.50 (0.08 to 0.92; p < 0.05) (Figure 15).

Discussion
The main aim of the meta-analysis was to examine the effect of multimodal rehabilitation on cytokine levels and other predominant variables in patients with FM.The intervention by Ernberg et al. [20] significantly decreased the scores of the FIQ in favor of the relaxation group, but the two fibromyalgia groups examined did not find functional and clinical differences in the change in values from pre to post-test.Other studies showed differences in variables in favor of the control group in pain ratios applying programs of 15 weeks of strength exercise 2 sessions/week 1 session/60 min 10 min warm-up 50 min and lower limb strength, however, this intervention did not normalize a chronic inflammation profile nor did it have any effect on the anti-inflammatory effect in patients with FM symptoms on the clinical and functional variables examined [17,20,21].
Furthermore, the response of cytokine levels to intervention was found to differ between the groups.While in the healthy group, cytokine levels are low and increase during the intervention, in the experimental group, cytokine levels are elevated and decrease during the intervention.This observation suggests the presence of chronic inflammation in FM patients, which supports the hypothesis that inflammation and altered immune

Discussion
The main aim of the meta-analysis was to examine the effect of multimodal rehabilitation on cytokine levels and other predominant variables in patients with FM.The intervention by Ernberg et al. [20] significantly decreased the scores of the FIQ in favor of the relaxation group, but the two fibromyalgia groups examined did not find functional and clinical differences in the change in values from pre to post-test.Other studies showed differences in variables in favor of the control group in pain ratios applying programs of 15 weeks of strength exercise 2 sessions/week 1 session/60 min 10 min warm-up 50 min and lower limb strength, however, this intervention did not normalize a chronic inflammation profile nor did it have any effect on the anti-inflammatory effect in patients with FM symptoms on the clinical and functional variables examined [17,20,21].
Furthermore, the response of cytokine levels to intervention was found to differ between the groups.While in the healthy group, cytokine levels are low and increase during the intervention, in the experimental group, cytokine levels are elevated and decrease during the intervention.This observation suggests the presence of chronic inflammation in FM patients, which supports the hypothesis that inflammation and altered immune response may play a role in the development and maintenance of FM symptoms [4].In addition, it also provides a possible explanation for many of the symptoms experienced by FM patients.For example, elevated concentrations of IL-6 and IL-8 have been shown to have additive or synergistic effects on the perpetuation of chronic pain in these patients [30].IL-6 induces pain, fatigue, and psychiatric disorders such as depression and stress [4], while IL-8 is associated with pain and sleep disorders [16].Consequently, an imbalance between proinflammatory and anti-inflammatory cytokines contributes to chronic peripheral sensitization of the nervous system and pain processing [2,4].Another study also points to an imbalance between proinflammatory and anti-inflammatory cytokines, with higher levels of proinflammatory cytokines such as TNF-α, IL-1ra, IL-6, and IL-8 [6].These results support the hypothesis that inflammation and immune responses are altered in the FM group.
Although the heterogeneity associated with the processes, techniques, and instruments for the detection of cytokines in different body fluids represents an underlying factor in the literature, we highlight the importance of using the mean difference technique.standardized in the meta-analysis.The study had difficulties in making Forrest plots regarding cytokines and the correlation with exercise in the study, as the authors did not show sufficient data to calculate the effect size from the original individual trial data.Our work showed a high methodological and statistical heterogeneity of the studies.The lack of data made it impossible to analyze all the variables, which is a limitation when it comes to quantifying the effects.This could affect the results of the meta-analysis, as some effects found were negligible in the meta-analytic model.However, a qualitative description of the effects of these variables has been made.
Proinflammatory cytokines are products of lymphocytes that are activated not only by injury but also by glial and neuronal cells.The main proinflammatory cytokines include IL-1, IL-2, IL-6, IL-8, IL-12, TNF-α, INF-α and IFN-α, while the main anti-inflammatory cytokines are IL-4, IL-10, IL-13, and TGF-α [4].In the studies reviewed, mainly 10 cy-tokines were analyzed, with IL-1β, IL-6, IL-8, and TNF-α standing out as proinflammatory cytokines.However, there was a lack of investigation and analysis of anti-inflammatory cytokines, both at the muscle level [17,18], which only investigated the levels of proinflammatory cytokines, and at the plasma level where, for example, IL-13 was not analyzed in any of the studies.
According to investigate the cytokines present in the muscle tissue and plasma of patients with FM.In the present systematic review, only 25% of the studies [17,18] employed the micro-dialysis technique in muscle tissue and revealed no significant difference in the release of inflammatory cytokines between the groups in terms of IL-1β, IL-6, IL-8.However, an increase in the cytokine TNF-α was observed only in the control group.These findings align with the conclusions of other studies suggesting that physical exercise affects the metabolic profile of muscle but does not impact the plasma immune profile of FM patients [17,22].
The final specific objective was to examine the physical therapy tools used to investigate the effects of such interventions on cytokine levels in FM patients.In the studies that evaluated the effects of exercise on cytokines, variable results were found.Regarding the study of anti-inflammatory cytokines, we identified that in three studies, no analysis of these cytokines was performed [15,17,18].In the other trials where they were analyzed, three found no evidence of an anti-inflammatory effect after the intervention, with no significant changes observed [20][21][22].However, in three other studies, there was an antiinflammatory effect [15,16,19].Significantly, this effect was not directly attributed to the action of anti-inflammatory cytokines but to the reduction of pro-inflammatory cytokines, suggesting a possible inhibition of some anti-inflammatory cytokines, such as IL-10 [20].For example, in one of the studies, after a moderate cycling session, an anti-inflammatory effect was found due to a slight decrease in IL-10 in the experimental group, but what made the difference was a significant reduction in IL-8 levels, attributed to a lower release of pro-inflammatory cytokines by monocytes and lower activation of neutrophils [16].In another study, a combination of exercise and infrared therapy was used, and a reduction in IL-6 levels was observed, exerting an anti-inflammatory effect, and improving pain and quality of life in FM subjects [19].However, after a 15-week strength exercise intervention, no anti-inflammatory effect was found on any FM symptoms [21].From the methodological point of view of the meta-analysis, only a few studies could be analyzed.When the data distribution is symmetrical, the median can be used in meta-analyses.This criterion was adopted methodologically with the studies by Enberg et al. [17,20], so it would have been indicated to meta-analyze these studies using the probability of superiority (PS) as an index of effect size given the non-parametric analysis used (Mann-Whitney U test) [31,32].Future lines of research could aim to carry out an analysis of the real impact of the size of the effect that is unknown by calculating the non-overlapping percentage of the population in order not to adopt risks.Likewise, in future research, it would be interesting to carry out a meta-regression that considered cytokines and the MFI (0-20) fatigue scale [33].
An important point of discussion that could be taken up in the future would be the probable association of virus infection with cytokines levels or immune responses in patients with FM.However, among the interests of the present study was to analyze the presence of anti-inflammatory and proinflammatory cytokines, but not to relate it to the probable viral association, since this hypothesis has been dismissed on some occasions.For example, what could be analyzed in the future is the possible relationship of FM symptoms with COVID-19 symptoms opening new etiopathogenetic horizons, but this objective will be pursued later.
The type of exercise, its duration, and intensity are also essential factors in the results obtained.For example, in a study of warm water aquatic exercise performed for eight months, two sessions per week, and 60 min per session, it was observed that after four months no anti-inflammatory effects were obtained.However, after eight months they were [15].Although we know that all patients with chronic pain and FM have lowgrade systemic inflammation, our meta-analysis was unable to demonstrate that blood biomarkers are specific or diagnostic for FM.The fact of having few studies and being very heterogeneous among themselves, does not provide sufficient scientific evidence to establish the cause-effect relationship of the treatments on the levels of cytokines and FM, for all this new research that analyzes the heterogeneity and seeks to carry out synthesis of results in a homogeneous way will be able to demonstrate the effect of the type of intervention carried out.In this case, it would be convenient to explore the moderate effect related to the participants and the characteristics of the intervention using regression techniques with confounding outcomes [11].
In another study of aquatic exercise in a heated pool, which lasted six weeks, three times per week and 50 min per session, together with thermotherapy, a decrease in IL-6 levels and improvements in pain and quality of life were observed in the FM group [19].According to future research lines, we recommended examining continuous covariates that could be subjected to individual and pooled meta-regression in a random-effects meta-regression model taking into account the mean age of the women with FM, weight, height, study durations (weeks or months), sessions, days a week, number of exercises or treatment content, and even the method of blood extraction or cytokine measurement as predictor variables.Subgroups could also be used and analyzed for effects for categorical variables (women with fibromyalgia and healthy controls).
Therefore, although it was the same type of exercise, differences were observed in the duration of the intervention, the number of sessions per week, the duration of each session, and the addition of another physiotherapy tool.

Conclusions
In this systematic review, we found evidence to support elevated levels of proinflammatory and anti-inflammatory cytokines in patients with fibromyalgia compared to healthy subjects.These findings suggest the existence of chronic inflammation that may be responsible for an altered immune response and play a role in fibromyalgia symptoms, as well as developmental and nervous system sensitization to chronic pain.The most studied and analyzed cytokines were IL-1β, IL-6, IL-8, and TNF-α, while there was a lack of investigation of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13.It was found through microanalyses that exercise affects the metabolic profile of muscle tissue but has no significant effect on the immune profile in the plasma of FM patients.Furthermore, results on the impact of exercise on cytokine levels have been variable, so it is essential to consider factors such as type of exercise, duration, intensity, and combination with other physiotherapeutic tools.These aspects are fundamental to formulating recommendations and intervention strategies for managing FM.However, our meta-analysis has reported a state-of-the-art, but the reported evidence is very low due to problems with the study design, the small number of participants, and the low certainty of the results after the effects of physiotherapy on cytokine levels.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines11092367/s1,Table S1: Descriptive table; Table S2: Studied cytokines; Table S3: Analysis of the studied cytokines in groups of healthy subjects and subjects with FM; Table S4: Rick of bias assessment.

Figure 1 .
Figure 1.Flow diagram according to the PRISMA declaration.

Figure 1 .
Figure 1.Flow diagram according to the PRISMA declaration.

Figure 5 .
Figure 5. Effects of the treatment compared to the comparison group on the Algometry (PPT) in the left and right limb [20,21].

Figure 5 .
Figure 5. Effects of the treatment compared to the comparison group on the Algometry (PPT) in the left and right limb [20,21].

Figure 5 .
Figure 5. Effects of the treatment compared to the comparison group on the Algometry (PPT) in the left and right limb [20,21].

Figure 8 .
Figure 8. Effects of treatment versus comparison group on Pain (0-100) [20,21].Regarding the effects of treatment versus comparison group on Static strength elbow flexion force, no statistically significant changes were found in the combination of values from pre to post-test in the results presented by right and left in any of the studies examined (p > 0.05) (Figures 9 and 10). ).

Figure 9 .
Figure 9. Effects of treatment versus comparison group on Static strength elbow flexion force (Kg)-Presented by right and left [17,20].

Figure 8 .
Figure 8. Effects of treatment versus comparison group on Pain (0-100) [20,21].Regarding the effects of treatment versus comparison group on Static strength elbow flexion force, no statistically significant changes were found in the combination of values from pre to post-test in the results presented by right and left in any of the studies examined (p > 0.05) (Figures 9 and 10). ).

Figure 8 .
Figure 8. Effects of treatment versus comparison group on Pain (0-100) [20,21].Regarding the effects of treatment versus comparison group on Static strength elbow flexion force, no statistically significant changes were found in the combination of values from pre to post-test in the results presented by right and left in any of the studies examined (p > 0.05) (Figures 9 and 10). ).

Figure 9 .
Figure 9. Effects of treatment versus comparison group on Static strength elbow flexion force (Kg)-Presented by right and left [17,20].Figure 9. Effects of treatment versus comparison group on Static strength elbow flexion force (Kg)-Presented by right and left [17,20].

Figure 9 . 19 Figure 10 .
Figure 9. Effects of treatment versus comparison group on Static strength elbow flexion force (Kg)-Presented by right and left [17,20].Figure 9. Effects of treatment versus comparison group on Static strength elbow flexion force (Kg)-Presented by right and left [17,20].Biomedicines 2023, 11, x FOR PEER REVIEW 13 of 19

Figure 10 .
Figure 10.Effects of treatment versus comparison group on Static strength knee extension force (N)-Presented by right and left [17,20].

Figure 10 .
Figure 10.Effects of treatment versus comparison group on Static strength knee extension force (N)-Presented by right and left [17,20].

Figure 14 .
Figure 14.Effects of treatment versus comparison on IL-1β (pg/mL).b: second measurement of this outcome in the original study[17,19].

Figure 14 .
Figure 14.Effects of treatment versus comparison on IL-1β (pg/mL).b: second measurement of this outcome in the original study[17,19].

Figure 14 .
Figure 14.Effects of treatment versus comparison on IL-1β (pg/mL).b: second measurement of this outcome in the original study[17,19].

Figure 14 .
Figure 14.Effects of treatment versus comparison on IL-1β (pg/mL).b: second measurement of this outcome in the original study[17,19].

Figure 15 .
Figure 15.Effects of treatment versus comparison on IL-6 (pg/mL).b: second measurement of this outcome in the original study.b: second measurement of this outcome in the original study [17-19,22].

Figure 15 .
Figure 15.Effects of treatment versus comparison on IL-6 (pg/mL).b: second measurement of this outcome in the original study.b: second measurement of this outcome in the original study [17-19,22].
1: elegibility criteria were specified; 2: subjects were ramdomly allocated to groups; 3: allocation was concealed; 4: the groups were similar at baseline regarding the most important prognostic indicators; 5: blinding of all subjects; 6: blinding of all therapist who administered the therapy; 7: blinding of all assessors who measured at least one key outcome; 8: >85% outcomes of the subjets initially allocated to groups; 9: data for at least one key outcome by "intention to treat"; 10: between-group statistical comparisons; 11: point measures and measures of variability; N/A: not available.