Effectiveness of acute L-arginine supplementation on physical performance in strength training: a systematic review and meta-analysis [version 1; peer review: awaiting peer review]

Background: The oral administration of L-arginine has been related to improved physical performance due to a likely reduction in muscle fatigue, resulting from the vasodilator effect of nitric oxide on skeletal muscle. However, there is no precise and quantitative analysis of the information in the literature. The main objective of this study was to assess the effectiveness of L-arginine supplementation on physical performance in strength training with a systematic review and metaanalysis. We hypothesized that L-arginine supplementation would improve performance capacity and the effects involved in strength training. Methods: The study period was from 2010 to 2020. The inclusion process established articles with well-designed human experiments that included only L-arginine supplementation (without any additional compounds) testing the effects of L-arginine supplementation on sports performance related to strength training; identical experimental conditions in placebo or control group; and publications in the last ten years (until December 31, 2020). Three studies were included that compared L-arginine supplementation with placebo in anaerobic performance tests. Test analysis supplementation with other supplements was removed and there was no gender, age, and ethnicity level. Results: There was no significant heterogeneity (p>0.05) in the analysis of the three selected articles and the effects of L-arginine supplementation in muscular endurance; performance had a mean of 0.26 (95% CI = -0.129; 0.649; p = 0.190), peak torque with a mean of 0.002 (95% CI = -0.531; 0.527; p = 0. 99) of the third series of exercises and, furthermore, when comparing the integrated effect (resistance rate with the peak torque) there was no difference with a mean of Open Peer Review Reviewer Status AWAITING PEER REVIEW Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 13 F1000Research 2021, 10:1072 Last updated: 22 OCT 2021


Introduction
Athletes and recreational resistance exercisers have commonly associated training with nutritional supplementation to improve sports performance, maximize muscle mass and strength gains. 1,2 Proteins and amino acids represent 35 to 40% of the ergogenic resources used by this population. 3 In this context, supplementation with L-arginine, classified as a nonessential amino acid, has been advocated for resistance training practitioners for different reasons. 4 First, for its potential to promote vasodilation and supply energy to active muscles, given its participation in the synthesis and availability of nitric oxide (NO). 5 Second, increasing the creatinine concentration in the skeletal striated muscle promotes improved physical performance and decreased muscle fatigue. 6 Third, by promoting an increase in growth hormone production, known as GH (growth hormone), that stimulates protein synthesis and, consequently, muscle mass. 4 Despite being highly popular among exercise practitioners, few studies have examined the effect of oral administration of L-arginine on the ability to improve physical performance during resistance training, nor its role in increasing lean mass and maximizing gains force. 5 In addition, studies on the subjects used different protocols, dosages, populations, and associations with other compounds, presenting contradictory outcomes, making it challenging to make decisions about the use of L-arginine and drawing conclusions about the efficacy and safety of L-arginine supplementation.
Recently, Ojeda et al. 7 and Viribay et al. 8 performed studies of a systematic review with and without meta-analysis, respectively, on the effects of L-arginine on the physical performance of strength training practitioners. For the outcomes observed in the studies by Ojeda et al. 7 in respect to short-term supplementation and doses ranging from 0.075 to 8.0 gÁd À1 , it seemed to not influence variables such as nitrogen oxide (NOx), muscle oxygenation, oxygen consumption, and growth hormone secretion. The long-term outcomes, tested from three days to six weeks with maximum doses of 12 gÁd À1 , showed no effect and NO production during intermittent anaerobic exercise, nor in hemodynamic and vascular responses, maximal oxygen uptake (VO 2 max), insulin, cortisol, growth hormone or insulin type 1 growth factor (IGF-1) in resistance exercise or strength. However, at the end of the research, there was an investigation that reported a significant effect of prolonged L-arg supplementation on exercise performance. Evidencing a faster recovery from muscle injuries caused by decreased levels of the enzyme lactate dehydrogenase (LDH) after training, and a positive impact on anaerobic performance, mainly due to the acceleration of muscle recovery processes. Both outcomes were performed with men and women in anaerobic and aerobic activities. For Viribay et al. 8 the effective dose of L-arginine supplementation in acute protocols should be adjusted to 0.15 g/kg (10-11 g) ingested 60-90 min before exercise to improve aerobic and anaerobic activities performed by men and women. Based on these findings, the authors indicated that most studies with L-arginine are not favorable to its use as a nutritional ergogenic resource and point to the need for further investigation on the subject. The recent recommendation of the International Society for Sports Nutrition draws attention to this 5 by indicating that L-arginine supplementation has a little scientific basis to ensure its safety and nutritional efficacy.
There is no clear and quantitative information analysis of the different effects involved in the anaerobic performance process, nor studies that use only the substance L-arginine, without adding any other compound, and this may be necessary to better understand the efficacy, dosage, and time of ingestion. Therefore, we cannot assume that some positive performance results are due only to L-arginine, as the association with other compounds simultaneously can mask the true and real effect since none of these protocols investigated the underlying mechanisms and/or not use of only the compost L-arginine 6,9,10 and that the samples are expanded when there is an association of the supplement with other compounds. This has a confounding factor in the efficacy of the substance L-arginine. We hypothesized that L-arginine supplementation would improve performance capacity and the effects involved in anaerobic activities.
Given the above, this study intends to advance with current knowledge by evaluating the effectiveness of L-arginine in the physical performance of resistance training practitioners from a systematic review and meta-analysis. Conducting a systematic review and meta-analysis on the effects of L-arginine supplementation in resistance exercise practitioners will contribute to current knowledge by i) analyzing the effects of supplementation on physical performance; ii) evaluate the effectiveness of exclusive L-arginine supplementation in healthy and physically active populations; iii) investigate the dose-response relationship of oral L-arginine consumption.

Methods
This systematic review and meta-analysis were performed according to PRISMA ® (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) PICOS declaration guidelines and methodology, which defines the "Population," "Intervention," "Comparisons," "Results," and "Study Design." The review research was carried out considering the following phases: The identification of the article selection process in the first phase was carried out in January 2021 in the Cochrane, PubMed, MEDLINE, LILACS, and SciELO databases. For the selection of keywords, the descriptors in Health Sciences-DeCS were used, obtained through advanced searches in the union of search terms, including the "Mesh." The descriptors used were: "L-arginine"; "resistance exercises" and "strength training," a cognitive logical operator "AND," and "NOT," were used for the effective tracking of publications.
The inclusion process established that articles with the following characteristics would be used: (1)  Texts with the following characteristics were excluded: (1) L-arginine supplementation with some other compound; (2) investigations in which the topic was not essentially the use of L-arginine; (3) different samples of the effects of using the L-arginine not mentioning its effects (articles that do not show L-arginine results, in particular); (4) data from studies without Qualis status and surveys that did not show results; (5) other types of outcomes exclusively from reviews, pneumatic, contagious, cancerous and transmissible diseases.
Two authors independently screened and agreed with the selected studies for eligibility (CP, ROG, and TRL). Likewise, after the inclusion/exclusion criteria are applied to each study, the source study data (including authors and year of publication), sample size, participant characteristics (level, race, and gender), supplement administration (dose and time), and final results of interventions were extracted independently (CP and ROG) using an Excel spreadsheet (Microsoft Inc, Seattle). Then, possible disagreements were resolved through discussion until a consensus was reached, or by third-party judgment (MJK). In this sense, the Kappa coefficient, which indicates the reliability between raters, among the authors was above 90 with a level of agreement "almost perfect". 11 The Assessment of Multiple Systematic Reviews (AMSTAR2) tool was applied in the systematic review to assess the methodological quality of the studies. 12 The reviewers searched the studies for data that answered each question, and each reviewer's final ratings were placed on a spreadsheet to discuss agreements and disagreements, with a score assigned to each assessed study. To assess the quality of the evidence and the strength of the recommendations, the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system was used, 13 in which the research question was structured considering the domains of the acronym PICOS.
According to the Cochrane Collaboration Guidelines, to assess the risk of bias of randomized clinical trials, the RoB 2.0 tool (Risk of Bias -Cochrane risk of bias tool revised for randomized trials) was used and evaluated by two authors independently, and disagreements were resolved by discussion and/or a third author. The list was separated into six different domains: selection bias (random sequence generation, allocation concealment); performance bias (blinding of participants and researchers); detection bias (masking the outcome assessment); friction bias (incomplete result data); reporting bias (selective reporting); and other sources of bias. 14 Domains were considered as 'low' if the criteria met a low risk of bias (probable bias that would not seriously alter the results) or 'high' if the criteria presented a high risk of bias (probable bias that seriously weakens confidence in the results), or it was considered "not clear" (a plausible bias that raises some doubts about the results), whether the risk of bias was unknown. Full details of each article and domain (Figures 1 and 2).
The Clinical Trials database was queried using the terms "descriptor" and "descriptors" to locate potential studies with L-arginine. Phase 2, 3, and 4 clinical studies enrolled in the aforementioned database were considered. For this review, approval by the ethics committee was not required due to the use of secondary data.

Statistical analyses
The meta-analysis was performed using the Comprehensive Meta-Analysis (CMA) software, version 3.3.070, including the three selected articles, one for each exercise series (first, second and third). The effect size was calculated based on the difference in performance between groups L-arginine and control group. We calculated the effect standardized by the standard deviation (SMD), since the variables were not presented in the same unit in all studies, for example, total work of the series and total work presented in Joule, peak torque in Nanometer, number of maximum repetitions and rate of resistance).
Some studies presented performance measures for more than one muscle group, each subgroup of these studies was included in the analysis as if it were a different study. To test the effect of L-arginine with no sample overlap, combined analyzes with all subgroups from each study were also presented and included in the analysis as a single effect per study. Sensitivity analyzes were also performed for strength endurance measurements (total set work and total work, peak torque, maximum repetition number, and endurance rate) and peak torque (Nanometer). In the case of peak torque, as all studies presented data in the same measurement unit (Nanometer), we present the data in gross mean difference.
The inconsistency statistic (I 2 ) was calculated as an indicator of the percentage of the total variation observed within the studies due to real heterogeneity rather than chance, to avoid errors when using the Q statistic in the evaluation of heterogeneity. 14 I 2 values were included from 0 to 100%, representing a small amount of inconsistency between 25% and 50%, the average amount of heterogeneity between 50% and 75%, and a large amount of heterogeneity when the I 2 value  was greater than 75%. 15 In this sense, low, moderate and high adjectives would be accepted referring to I 2 values of 25%, 50%, and 75%, respectively, although a restrictive categorization was not appropriate in all circumstances. 15,16 Publication bias was analyzed using the Egger test and a p-value ≤ 0.05 was considered significant. Data were presented as standardized mean difference and 95% confidence interval, and we considered significant p ≤ 0.05.

Study identification
The studies that met the criteria to be included in this review are shown in the flowchart (Figure 3). The bibliographic search through electronic search identified a total of 1075 records according to the selected research equation, with 402 duplicate records being removed and 233 records removed for presenting animal tests, resulting in 440 records. Thus, after screening a total of 437 records were excluded from which: 52 were review studies, four inadequate results (use of the supplement without performing the physical activity), 123 performed on animals, 166 included more than one supplement, 83 did not use the substance L-arginine from the selected studies was removed from the quantitative synthesis and thus a total of three studies (n = 3) were included in the analysis. [17][18][19] In addition, the test of Egger (p > 0.3) and it was found that there was no significant effect for the risk of publication bias in the meta-analysis performed ( Figure 4).

Characteristics of the studies
The publication period of the three studies ranged between 2012 and 2018 and the sample size ranged from 12 to 20 individuals. The average age ranged from 27 to 70 years, with two studies with a male population and one female, that is, in total the population represented was 57.44% male and 42.55% female. All studies were conducted in a randomized, double-blind, placebo-controlled, one-day (acute) period of intervention. The amount of L-arginine used was 6 g 17,19 and 8 g 18 per individual. Tests were performed on the upper limb, 17 bottom, 18 and upper and lower. 19 According to Table 1,  the study by Meirelles and Matsuura 19 aimed to evaluate the acute effect of supplementation with L-arginine in the performance of strength in biceps flexion in three sets, evaluating the total work set, total, peak torque, and resistance rate. The study by Aguiar et al. 18 evaluated muscle performance in supplementation by measuring isokinetic strength data of the knee extensor and flexor muscles. And Alvares et al., 17 analyzed the maximum number of repetitions of the lower and upper limbs in three sets.

Quality of evidence
The Quality of the evidence generated by the present study was assessed by the GRADE system, considering the assessment items suggested for clinical trials. 13 The risk of bias in the primary studies included was almost non-existent, as they used crossover designs, with a control group, double-blind, with secret and randomized allocations, with a oneweek wash-out period.
There was no individual influence of any specific study on the summarized effects, with high homogeneity, low inconsistency (I 2 = 0.000%), in addition to the methodological similarity between the studies. The evidence was completely direct (well-defined cause and effect relationship) and no publication bias was observed, neither qualitatively through funnel graph analysis nor even in the hypothesis test (Egger). The only item that reduced the quality of evidence in this analysis was the low sample size (n = 30), however, there was still good precision, therefore, the level of evidence was identified as moderate (GRADE score = 3) for the effect gives L-arginine in strength performance.
Effect of L-arginine on performance: a meta-analysis Figure 5 shows that L-arginine had no significant effect on physical performance. There was no significant heterogeneity (p > 0.05) in any analysis of the three selected articles and, therefore, we used fixed effects in all analyses. The effects of L-arginine in the performance in muscular endurance it had an average of 0.26 (95% CI = À0.129; 0.649; p = 0.190), mean peak torque of À0.002 (95% CI = À0.531; 0.527; p = 0.99) of the third set of exercises and in addition, when comparing the integrated effect of resistance rate with peak torque, there was no significant difference with an average of 0.168 (95% CI = À0.145; 0.481; p = 0.292) ( Figure 5).
The meta-analysis reported a small amount of inconsistency between the integrated effect studies reviewed (I 2 = 0%; p = 0.99). The three analyzed studies and their subgroups had no positive effect on performance outcomes after L-arginine supplementation. In summary, there was no significant effect of L-arginine on performance under any condition tested.
Based on the analyses performed, it is inferred that L-arginine does not improve performance in any of the sets, in the measures of maximum strength or strength endurance, nor when the subgroups of the studies were combined in a single effect (integrated effect).

Discussion
This systematic review and meta-analysis aimed to assess the effectiveness of L-arginine on acute strength training performance. Our findings indicate that there was no significant effect of L-arginine on the production of maximum force and the endurance of force. Regarding dosage, differences of 6 and 8 g/day are observed, suggesting that both strategies may have similar physiological responses. Finally, L-arginine supplementation ingested 60-80 minutes before acute exercise (acute protocol) did not demonstrate performance improvement in strength training. It is known that the L-arginine is related to NO production and increased blood flow, contributing to vasodilation and possibly an increase in performance in various activities. 20 However, of the three studies analyzed in our systematic review and meta-analysis, only the study by Álvares et al., 17 observed increased blood flow with L-arginine supplementation. Furthermore, no study observed any difference in the concentrations of NO precursors with L-arginine supplementation. These findings justify why the three studies included in our review and meta-analysis did not demonstrate the ergogenic effects of L-arginine supplementation on strength training performance. As an example, Meirelles and Matsuura 21 investigated the effects of L-arginine supplementation on the number of maximum repetitions in the bench press (three sets of 70% of one-repetition maximum ) and the leg extensor (three sets of 80% of 1-RM) in 12 physically active men. After the evaluations, the researchers observed no improvement in performance or changes in the concentrations of NO precursors with L-arginine supplementation.
The studies analyzed in this meta-analysis had no effect of supplementation of L-arginine on performance in strength training and was associated with lack of improvement in performance in the group L-arginine compared to the placebo group to the analyzed subgroups (extension exercises, flexion of lower and upper limbs together with the number of repetitions) of resistance rate and peak torque. Furthermore, not even the integrated effect was observed as a significant result when it contains an overlapping effect. However, our findings are not in line with other studies that observed increases in anaerobic performance 22,23 and aerobic 24  Evidence indicates that L-arginine combined with alpha-ketoglutarate can improve performance in some activities, due to the increased production of adenosine triphosphate through the electron transport chain. 28 Specifically, alpha-ketoglutarate is a metabolite produced by the oxidative decarboxylation of isocitrate; a process that occurs in the Krebs cycle. 26,29 An exogenous supply of alpha-ketoglutarate through supplementation of L-arginine alpha-ketoglutarate (AAKG) can increase Krebs cycle flux, increasing the oxidation rate of acetyl-CoA. 28 In the study by Bailey et al., 9 supplementation was carried out with L-arginine in association with maltodextrin (supplementation also used in the study by Liu et al., 30 found in the meta-analysis by Ojeda et al. 7 ). The possibility of reducing the rate of glycogen degradation or even increasing this content can help to reduce fatigue and thus support the capacity for exercise intensity without exhaustion since there is a link between the glycogen content muscle fiber and its ability to perform intense repeated contractions. 31,32 Muscles have complex carbohydrate stores made up of glucose molecules called glycogen, and the ingestion of maltodextrin, or any other carbohydrate, causes a decrease in the breakdown of liquid glycogen during long-term exercise. 33,34 This process maintains carbohydrate oxidation throughout the body and energy production, which can occur in both men and women. 35 The combination of this element together with proteins and/or amino acids can promote glycogen increase, recovery, and stimulate muscle protein synthesis after an intense exercise protocol. 28,31,36 As for strength exercises, for post-exercise muscle recovery of glycogen, carbohydrate combined with protein offer a greater contribution to sports performance, increasing the time for exhaustion, muscle strength, and muscle power. 37,38 The possibility of reducing the rate of glycogen degradation or even increasing this content can help to reduce fatigue and thus support the capacity for exercise intensity without exhaustion since there is a link between the glycogen content muscle fiber and its ability to perform intense repeated contractions. 31,32 Muscles have complex carbohydrate stores made up of glucose molecules called glycogen, and the ingestion of maltodextrin, or any other carbohydrate, causes a decrease in the breakdown of liquid glycogen during long-term exercise. This process maintains carbohydrate oxidation throughout the body and energy production, which can occur in both men and women. The combination of this element together with proteins and/or amino acids can promote glycogen increase, recovery, and stimulate muscle protein synthesis after an intense exercise protocol. 28,31,36 As for strength exercises, for post-exercise muscle recovery of glycogen, carbohydrate combined with protein offer a greater contribution to sports performance, increasing the time for exhaustion, muscle strength, and muscle power. 37,38 For this systematic review and meta-analysis, all included studies used only L-arginine without adding other supplements, which limits the possibility of obtaining synergistic or antagonistic effects in conjunction with other compounds.
Studies confirm that L-arginine is related to NO synthesis and also its role as a cell-signaling molecule. However, due to the rapid oxidation by oxyhemoglobin to nitrate and nitrite, a fact that contributes to the half-life of the blood is greatly reduced and the quantification of NO production becomes more difficult. 39 According to current literature, there are some obstacles about protocols, doses, and supplementation time, which are controversial due to the results around L-arginine supplementation and its effects. 40 This is the first meta-analysis investigating the acute effects of L-arginine supplementation on strength training performance. As for dosages and time of ingestion, due to the various current methods, it is suggested that more research should be carried out in the homogenization of L-arginine supplementation protocols regarding dosages and time of ingestion. Statistically, the number of studies (n = 03) analyzed in this systematic review and meta-analysis and the low sample number of each selected article related to L-arginine supplementation presupposes to represent a limitation with risk of bias, making them more sensitive to identify differences. However, for this review, the selection and analysis of studies, interpretation, and quantification of results represent strengths when evaluated by the AMSTAR guidelines, where GRADE scores and analysis were classified as "high quality" 12 and "moderate", 13 respectively.
In the same vein, this meta-analysis was carried out with homogeneous statistics, which constitutes an important point in favor. The choice to select studies that did not include systematic reviews implies raising evidence for further studies to be carried out in favor of the result, which aims to demonstrate the evident effect of positive performance with the use of L-arginine in physical tests related to muscle performance.
The dosage of L-arginine supplementation used in the studies may be a reason why changes were not observed in the concentrations of NO precursors and the performance improvement in strength training. The dosage of L-arginine used in the studies analyzed in our meta-analysis ranged between 6 and 8 g/day. However, Viribay et al. suggested L-arginine intake for 0.15 g/kg of body weight after performing a meta-analysis to assess the acute effects of L-arginine supplementation on the performance of aerobic and anaerobic predominance activities. . This dosage suggested by Viribay et al. agrees with the three studies analyzed in our meta-analysis, in which participants were supplemented with 6-8 g/day of L-arginine. Furthermore, the ergogenic effects with L-arginine supplementation depend on the time of ingestion/dosage and the onset of the analyzed activity, as blood levels of nitrate and nitrite increase 2.5-3 hours after supplement ingestion. rich in nitrate, 41 and the three studies analyzed in our meta-analysis ingested L-arg 60-80 minutes before starting the evaluations.
According to the results obtained, new dosages of L-arginine supplementation should be proposed, as well as studying the differences between elite and amateur athletes. Finally, a new research perspective should be considered studying the L-arginine absorption capacity involving mechanisms in physical activity practitioners, as this may represent a crucial understanding of the knowledge of supplementation.

Final considerations
The supplementation of L-arginine does not provide an ergogenic effect on strength training performance. The effective dose of supplementation L-arginine, exercise protocols, and supplementation duration are not yet well understood and must be adjusted so that supposedly the anaerobic performance responses are positive. Because of this, some differences in supplementation and timing protocols were found that limited the drawing of robust conclusions, suggesting that published studies with meta-analysis lack credibility for this type of study. Thus, more clinical studies are needed to support systematic review and meta-analysis studies.
The publication bias obtained in studies of anaerobic performance means that practitioners should take these results with caution when applying the analyzed results to reality. Considering the importance of the theme and the sample size, the study has limitations due to the lack of bibliographic references expressed in the scientific world. Finally, we suggest further studies to compare the effects of L-arg supplementation without the association with other compounds on strength training performance, as the combination of compounds with L-arginine may mask the results of other studies analyzed.
However, this study should not be considered exhaustive and further research will be substantially important to strengthen future results. Such an effort is still needed and would pave the way for future studies with phytochemicals and pharmacology. For new scientific investments with this goal, we also recommend a more detailed investigation and one that fundamentally approaches the benefits of using L-arginine for human health. Finally, disseminating duly proven information about the use of L-arginine in practice will strengthen the values and richness of modern studies dealing with human health.

Registration and protocol
Review was not registered and protocol was not prepared.

Data availability
All data underlying the results are available as part of the article and no additional source data are required.

Reporting guidelines
Dryad: Effectiveness of acute L-arginine supplementation on physical performance in strength training: a systematic review and meta-analysis, https://doi.org/10.5061/dryad.2rbnzs7pd.