Effects of Omega-3 Supplementation Alone and Combined with Resistance Exercise on Skeletal Muscle in Older Adults: A Systematic Review and Meta-Analysis

Sarcopenia negatively affects skeletal muscle mass and function in older adults. Omega-3 (ω-3) fatty acid supplementation, with or without resistance exercise training (RET), is suggested to play a role as a therapeutic component to prevent or treat the negative effects of sarcopenia. A systematic review and meta-analysis were conducted on the impact of ω-3 fatty acid supplementation with or without RET on measures of muscle mass and function in older adults (≥55 y). The data sources included SPORTDiscus, PubMed, and Medline. All the study types involving ω-3 fatty acid supplementation on measures of muscle mass and function in older adults (without disease) were included. The mean differences (MDs) or standardized mean differences (SMDs) with 95% confidence intervals were calculated and pooled effects assessed. Sixteen studies (1660 females, 778 males) met our inclusion criteria and were included in the meta-analysis. ω-3 fatty acid supplementation did not impact lean tissue mass (SMD 0.09 [−0.10, 0.28]). Benefits were observed for lower body strength (SMD 0.54 [0.33, 0.75]), timed-up-and-go (MD 0.29 [0.23, 0.35]s), and 30-s sit-to-stand performance (MD 1.93 [1.59, 2.26] repetitions) but not walking performance (SMD −0.01 [−0.10, 0.07]) or upper body strength (SMD 0.05 [−0.04, 0.13]). Supplementing with ω-3 fatty acids may improve the lower-body strength and functionality in older adults.


Introduction
Sarcopenia, characterized by the age-related reduction in strength and muscle mass, is a global health issue [1]. A recent systematic review and meta-analysis found that sarcopenic older adults had reduced functionality and were at greater risk of experiencing a fall compared to non-sarcopenic adults [2]. The direct healthcare costs related to treating sarcopenia in the United States of America were $18.5 billion in 2000 [3], which is projected to increase to $30.5 billion in 2022 (inflation estimated with https://www.in2013dollars.com, accessed on 10 April 2022). This cost has undoubtedly risen even more due to the aging population, and it is suggested that more high-quality research utilizing cost-effective strategies to counteract these effects is warranted [3]. Further, several scientific studies have

Materials and Methods
This systematic review was completed in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement [25]. A literature search was conducted using PubMed, SPORTDiscus, and MedLine, including all dates from inception up to 7 May 2022. The following Boolean phrase was used: (omega-3 OR omega 3 OR n-3 OR fishoil OR fish oil OR EPA OR DHA OR ALA or eicosapentaenoic acid OR docosahexaenoic acid OR acid α-linolenic acid OR alpha-linolenic acid) AND (resistance training OR muscle function OR hypertrophy OR atrophy) AND (older adult OR elderly OR aging OR sarcopenia). No restriction on language or date was implemented. Articles meeting the following participants, intervention, comparators, outcomes, study type (PICOS) were included: Participants were adults ≥55 years old, without major disease conditions (for example, cancer, chronic obstructive pulmonary disease). The intervention of interest was supplemental fish oil or ω-3 (ALA, DHA, EPA) supplementation, with or without RET. Studies involving a multi-ingredient supplement (e.g., ω-3 plus whey protein or other nutritional supplements) were not included. Studies involving a control (placebo) substance or no supplement as a comparator were included. Outcomes of interest included lean tissue (i.e., muscle) mass, lower-and upper body strength, and measures of functional ability (i.e., walking performance, timed-up-and-go test, repeated sit-to-stand testing). Randomized controlled trials as well as prospective study types were included. Only published material was included. Reference lists of included articles were also searched for any other articles that may fit our inclusionary criteria based on the above PICOS parameters.
Titles, abstracts, and full texts were screened to determine eligibility by two researchers, with any conflict settled by a third researcher. Two researchers completed the risk of bias for each article using the revised Cochrane risk of bias tool [26] and a third researcher settled disagreements. Sensitivity analyses were performed by removing studies with a high risk of bias or publication bias (i.e., from funnel plots) to determine if they impacted the outcomes of the meta-analyses.
Data were extracted from included manuscripts as means and standard deviations for intervention and comparator groups. Meta-analyses were run using RevMan 5.3 software (Cochrane Community, London, UK) using fixed-effects models. Mean differences (MDs) and 95% confidence intervals (CIs) or standardized mean differences (SMDs; where measurement techniques on the same outcome varied between studies) between intervention and control groups were calculated. Heterogeneity was assessed using χ 2 and I 2 tests, where heterogeneity was indicated by χ 2 p-values ≤ 0.1 or I 2 test value > 75%. When sufficient data were available, forest plots were generated using pooled effects. All data were assessed using standard mean difference, with the exception of the timed-up-and-go (TUG) and sit-to-stand tests, both of which were assessed using mean difference as the tests were the same across studies for these two measures. Funnel plots were generated to determine whether there was publication bias.

Risk of Bias
Of the sixteen articles included, three had a high risk of bias [34,39,42], four had some concerns [27,28,37,41], and nine had a low risk of bias (Table 2).
Studies with a high risk of bias were rated as such in the domain of risk of bias in the "measurement of the outcome" due to the lack of blinding of investigators, which may impact the assessment of factors such as strength. Studies with some concerns were rated as such in the "selection of reported results" because it could not be determined whether the trial was analyzed according to a pre-specified plan (i.e., the study was not registered before recruitment).

Lean Tissue Mass
A forest plot of the effect of ω-3 fatty acids on lean tissue mass is shown in Figure 2. Five articles reported the effects of ω-3 fatty acid supplementation alone on lean tissue mass in older adults [31,32,34,39,40], involving 297 participants (n = 58 male, n = 239 female). No significant difference was observed between the ω-3 fatty acids and control (p = 0.50). Five articles investigate the impact of ω-3 fatty acids in addition to RET in older adults [28,30,32,36,38], involving 217 participants (52 males, 165 females). Cornish & Chilibeck [27] reported the male and female results separately; therefore, these were assessed individually in the forest plot ( Figure 2). No impact of ω-3 fatty acid supplementation in conjunction with RET was found for lean tissue mass (p = 0.48). No significant difference was observed when all the studies were taken together (with and without RET) (p = 0.33).

Lower Body Strength
Pooled effects of ω-3 fatty acid supplementation on lower body strength with and without RET are displayed in Figure 3. Three articles [31,33,40] (209 participants; n = 56 males, 153 females) investigated the effects of ω-3 fatty acid supplementation on lower body strength without RET. The pooled analysis indicated a significant positive impact of ω-3 without RET on lower body strength (p < 0.001). Seven articles reported the effects of ω-3 fatty acid supplementation in conjunction with RET on lower body strength [27,31,[35][36][37][38]42] (n = 288; 102 males, 186 females). Both Cornish & Chilibeck [27] and Da Boit et al. [38] reported male and female results independently and, thus, they are displayed as such in the pooled analysis ( Figure 3). Leg strength was significantly improved in the ω-3 group compared to the control (p = 0.005), although significant heterogeneity was present (I 2 = 86% p < 0.001). When the study with a high risk of bias [42] was removed, the significant finding was no longer present (SMD 0.22 (−0.08,0.51), p = 0.15). When considering all the studies (with and without RET) after the high-risk study was removed, there was still a significant effect favoring the ω-3 over the control for lower body strength (p < 0.001), and a subgroup difference was present (p = 0.03), with ω-3 supplementation being more effective in increasing lower body strength when taken on its own compared to when taken with RET.

Lower Body Strength
Pooled effects of ω-3 fatty acid supplementation on lower body strength with and without RET are displayed in Figure 3. Three articles [31,33,40] (209 participants; n = 56 males, 153 females) investigated the effects of ω-3 fatty acid supplementation on lower body strength without RET. The pooled analysis indicated a significant positive impact of ω-3 without RET on lower body strength (p < 0.001). Seven articles reported the effects of ω-3 fatty acid supplementation in conjunction with RET on lower body strength [27,31,[35][36][37][38]42] (n = 288; 102 males, 186 females). Both Cornish & Chilibeck [27] and Da Boit et al. [38] reported male and female results independently and, thus, they are displayed as such in the pooled analysis ( Figure 3). Leg strength was significantly improved in the ω-3 group compared to the control (p = 0.005), although significant heterogeneity was present (I 2 = 86% p < 0.001). When the study with a high risk of bias [42] was removed, the significant finding was no longer present (SMD 0.22 (−0.08,0.51), p = 0.15). When considering all the studies (with and without RET) after the high-risk study was removed, there was still a significant effect favoring the ω-3 over the control for lower body strength (p < 0.001), and a subgroup difference was present (p = 0.03), with ω-3 supplementation being more effective in increasing lower body strength when taken on its own compared to when taken with RET.

Lower Body Strength
Pooled effects of ω-3 fatty acid supplementation on lower body strength with and without RET are displayed in Figure 3. Three articles [31,33,40] (209 participants; n = 56 males, 153 females) investigated the effects of ω-3 fatty acid supplementation on lower body strength without RET. The pooled analysis indicated a significant positive impact of ω-3 without RET on lower body strength (p < 0.001). Seven articles reported the effects of ω-3 fatty acid supplementation in conjunction with RET on lower body strength [27,31,[35][36][37][38]42] (n = 288; 102 males, 186 females). Both Cornish & Chilibeck [27] and Da Boit et al. [38] reported male and female results independently and, thus, they are displayed as such in the pooled analysis ( Figure 3). Leg strength was significantly improved in the ω-3 group compared to the control (p = 0.005), although significant heterogeneity was present (I 2 = 86% p < 0.001). When the study with a high risk of bias [42] was removed, the significant finding was no longer present (SMD 0.22 (−0.08,0.51), p = 0.15). When considering all the studies (with and without RET) after the high-risk study was removed, there was still a significant effect favoring the ω-3 over the control for lower body strength (p < 0.001), and a subgroup difference was present (p = 0.03), with ω-3 supplementation being more effective in increasing lower body strength when taken on its own compared to when taken with RET.  [27,31,33,[35][36][37]40,42].
Two studies were identified to have publication bias from funnel plot analysis (i.e., they fell outside the funnel) [38,42]. When these were removed, the violation of heterogeneity was no longer present. With these removed, the findings for ω-3 combined with RET were not significant, but the overall findings of ω-3 supplementation (with and without RET) were still significant (p = 0.0007), and a significant subgroup difference was present, with ω-3 supplementation being superior when taken on its own when compared to taken in conjunction with RET (p = 0.005).

Functional Performance Tests
Six studies reported TUG results [28,34,36,37,39,42] (n = 197; 66 males, 131 females). Of these, only two did not include an RET intervention [34,39]; we, therefore, did not include a sub-group analysis to compare studies with and without RET. The pooled analyses indicated improved performance in TUG (i.e., reduced time) with ω-3 (p < 0.001) ( Figure 5). These findings remained even when studies with a high risk of bias [34,39] were removed.

Functional Performance Tests
Six studies reported TUG results [28,34,36,37,39,42] (n = 197; 66 males, 131 females). Of these, only two did not include an RET intervention [34,39]; we, therefore, did not include a sub-group analysis to compare studies with and without RET. The pooled analyses indicated improved performance in TUG (i.e., reduced time) with ω-3 (p < 0.001)  Figure 5). These findings remained even when studies with a high risk of bias [34,39] were removed. Note that all mean results entered for each study are reduction in seconds within groups [28,34,36,37,39,42].
Nutrients 2022, 14, x FOR PEER REVIEW 11 of 16 ( Figure 5). These findings remained even when studies with a high risk of bias [34,39] were removed. Note that all mean results entered for each study are reduction in seconds within groups [28,34,36,37,39,42].

Discussion
The most important finding of this systematic review and meta-analysis is that ω-3 supplementation does not impact lean tissue mass with or without RET in older adults but does improve lower body strength and lower body functional performance. Supplementation was marginally effective in studies without resistance training for improving upper body strength, but only after studies high in risk of bias or publication bias were removed from analysis. Supplementation appears to be effective when taken alone, but less so when taken in conjunction with RET for improving strength. No impacts were observed for walking speed. Our finding that ω-3 supplementation improves lower body strength and functional ability is important because lower body strength is preferentially affected by aging [43], and sarcopenic older adults suffer from reduced functional performance [2]. Tests such as the TUG and sit-to-stand test, which were improved with ω-3 supplementation in our meta-analyses, are important predictors of functional ability in older adults [44][45][46].
Our hypothesis that ω-3 supplementation would enhance lean tissue mass (i.e., muscle mass) was based on a variety of narrative reviews, which have speculated that ω-3 supplementation may enhance skeletal muscle anabolism [47][48][49]. There are several physiological mechanisms by which ω-3 supplementation might improve muscle mass: ω-3 supplementation may reduce inflammation [24], which might reduce the activation of pathways involved in protein degradation [21]. ω-3 supplementation activates signaling proteins involved in the activation of translation [32,50] and enhances protein synthesis in response to amino acids or insulin [50]. Other studies have failed to find evidence for ω-3 supplementation enhancement of protein synthesis; for example, ω-3 supplementation during resistance training does not lead to increased activation of satellite cells or incorporation of satellite cells into muscle fibres as new myonuclei [32].
An enhancement of muscular strength with ω-3 supplementation, as found in our meta-analysis, might occur in the absence of improvements in muscle mass if neural activation is improved. ω-3 fatty acids may be incorporated into nervous tissue cell membranes [51], and this may affect the neural activation of muscle. Further, ω-3 supplementation during resistance training programs in older adults improves the ability to recruit motor units and reduces electromechanical delay, indicating enhanced neural activation of muscle [39]. Changes such as reduced electromechanical delay (i.e., a reduction in the time from neural activation to production of muscular force) could explain some of the improvements in functional ability (i.e., the TUG test, number of sit-to-stand repetitions within 30 s) observed with ω-3 supplementation in our meta-analyses.
Given the irrefutable role of RET in the delay and attenuation of sarcopenia [52][53][54], RET should always be the cornerstone of both preventative and treatment plans for sarcopenia. However, with less than half of older adults meeting physical activity guidelines [55], other strategies to mitigate the effects of aging on muscle are of interest. A variety of nutritional supplements, such as creatine monohydrate, whey protein, and vitamin D, have been used in older adults to combat the development and progression of sarcopenia [21]. The results of the current meta-analysis indicate that ω-3 supplementation improves lower body strength, but to a lesser amount when combined with resistance training. The very high stimulus of resistance training for improving strength may mask the effects of a dietary supplement.
There are several limitations with the findings from our meta-analyses. Some of our results were affected by the removal of studies deemed to have a high risk of bias or publication bias. These studies contributed to heterogeneity across our meta-analyses. When studies with a high risk of bias or publication bias were removed, ω-3 supplementation was no longer effective for improving lower body strength when taken during resistance training programs. Likewise, when studies with a high risk of bias were removed, the significant impact of ω-3 supplementation for improving sit-to-stand performance was lost. On the other hand, when studies with a high risk of bias or publication bias were removed from the upper body strength meta-analysis, ω-3 supplementation was marginally effective for improving strength in studies without a resistance-training component. These findings indicate a need for clinical trials that have a lower risk of bias.

Conclusions
The current systematic review and meta-analysis outline the impact of ω-3 supplementation on muscular mass and function in older adults. Supplementation with ω-3 fatty acids may improve strength and functional ability in older adults but does not appear to influence lean body mass. Overall, more research on ω-3 fatty acid supplementation in skeletal muscle is a high priority considering the potential benefits these types of fatty acids may have in reducing sarcopenia and dynapenia. It is recommended that high-quality randomized controlled trials with larger sample sizes are needed to fully elucidate the effects of ω-3 fatty acid supplementation on skeletal muscle mass and skeletal muscle function in older adults at risk of sarcopenia. Further, future research should elucidate any differences in the source of ω-3 fatty acids, such as plant-based (flax), plankton, krill, and fish. Optimal dosing strategies for improving muscle mass and function are also an area for future research.