Effects of Arginine Supplementation on Athletic Performance Based on Energy Metabolism: A Systematic Review and Meta-Analysis

c oxide related ergogenic aids such as arginine (Arg) have shown to impact positively on sport performance through several physiological and metabolic mechanisms. However, research results have shown to be controversial. The great di erences regarding required metabolic pathways and physiological demands between aerobic and anaerobic sport disciplines could be the reasons. The aim of this systematic review and meta-analysis was to evaluate the e ects of Arg supplementation on aerobic ( VO2max) and anaerobic (>VO2max) performance. Likewise, to show the e ective dose and timing of this supplementation. A structured search was carried out in accordance with PRISMA ® (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement and PICOS guidelines in PubMed/MEDLINE, Web of Science (WOS), and Scopus databases from inception to January 2020. Eighteen studies were included which compare Arg supplementation with placebo in an identical situation and testing its e ects on aerobic and anaerobic performance tests. Trials analyzing supplementation with other supplements were removed and there was not athlete's level, gender, ethnicity, or age lters. The performed meta-analysis included 15 studies and random e ects model and pooled standardized mean di erences (SMD) were used according to Hedges' g. Results revealed that Arg supplementation could improve aerobic (SMD, 0.84; 95% CI, 0.12 to 1.56; magnitude of SMD (MSMD), large; I2, 89%;p=0.02) and anaerobic (SMD, 0.24; 95% CI, 0.05 to 0.43; MSMD, small; I2, 0%;p=0.01) performance

The performed meta-analysis included 15 studies and random e ects model and pooled standardized mean di erences (SMD) were used according to Hedges' g. Results revealed that Arg supplementation could improve aerobic (SMD, 0.84; 95% CI, 0.12 to 1.56; magnitude of SMD (MSMD), large; I2, 89%;p=0.02) and anaerobic (SMD, 0.24; 95% CI, 0.05 to 0.43; MSMD, small; I2, 0%;p=0.01) performance tests. In conclusion, acute Arg supplementation protocols to improve aerobic and anaerobic performance should be adjusted to 0.15 g/kg of body weight ingested between 60–90 min before. Moreover, chronic Arg supplementation should include 1.5–2 g/day for 4–7 weeks in order to improve aerobic performance, and 10–12 g/day for 8 weeks to enhance anaerobic performance. Keywords:aminoacids; ergogenic aids; physical performance; nitric oxide; aerobic; anaerobic 1. Introduction Athletes often turn to nutritional supplements in order to maintain health and maximize athletic performance [1]. Among them, proteins and amino acids represent the most consumed ergogenic aids, with a frequency of 35–40% [2]. However, the use of nutritional supplements with vasodilatory function are increasing considerably in the sport eld, given that there is strong evidence that its intake Nutrients2020,12, 1300; doi:10.3390 /nu12051300 /journal/nutrients

Nutrients2020,12, 1300 2 of 20 has a positive e ect on athletic performance [3–5]. In this sense, although nitrate and beetroot juice are the most studied vasodilatory supplements in this eld [6,7], arginine (Arg) is an amino acid that has shown a vasodilator e ect because it participates in the synthesis and bioavailability of nitric oxide (NO) [4,8]. For this reason, Arg supplementation has been used by athletes in order to obtain improvements in athletic performance [9–12]. Although Arg is a non-essential amino acid for adults because is absorbed through dietary proteins [13] and synthesized in the small intestine from proline, glutamate, and glutamine [8,14], some research has shown that supplementation could be bene cial to increase athletic performance [15]. The most relevant bene t of Arg is related to NO synthesis and its role as a cell signaling molecule with physiological relevant e ects [16]. NO has shown increased blood ow and improved muscle contraction, gas exchange, oxygen kinetics, and mitochondrial biogenesis [6,17]. Otherwise, Arg has also been shown to stimulate the release of growth hormone (GH) [18,19], which helps to promote cell growth and regulate the mobilization of fuels in the body that contributes to increase muscle mass and hypertrophy [20–22]. Moreover, Arg supplementation has presented a reduction of ammonia, lactate, fatty acids, and fat oxidation levels after exercise [23,24]. Likewise, Arg has shown an increase in glycerol post-exercise, with improved carbohydrate oxidation and oxygen e ciency [25,26], considering these potential bene ts in endurance sport performance. Thus, Arg has displayed e ects on di erent physiological and metabolic pathways that could improve athletic performance in both, endurance or “aerobic” and high intensity or “anaerobic” athletic performance [15]. Athletic performance in endurance sports, in which e orts commonly last 5 min or more and requires equal and/or less intensity than VO2max, is related to the capacity of circulatory and respiratory systems to supply fuel and resynthesize adenosine triphosphate (ATP) by oxidative metabolism [27,28]. Therefore, endurance performance is determined by maximal oxygen uptake (VO2max), ventilatory thresholds, and energy e ciency or economy [28,29]. In this sense, di erent Arg

last 5 min or more and requires equal and/or less intensity than VO2max, is related to the capacity of circulatory and respiratory systems to supply fuel and resynthesize adenosine triphosphate (ATP) by oxidative metabolism [27,28]. Therefore, endurance performance is determined by maximal oxygen uptake (VO2max), ventilatory thresholds, and energy e ciency or economy [28,29]. In this sense, di erent Arg supplementation protocols (<7 days or acute) and dosages (6–10 g/day) have been shown to improve several physiological parameters and performance outcomes, such as time to exhaustion, mean power output, and exercise capacity in moderate-submaximal intensities [10,11,30]. These results could be explained only due to improvements in blood ow and oxygen supply to the muscles, because it seemed that longer supplementation periods are needed to enhance mitochondrial respiration and oxidative phosphorylation through NO pathway [6]. However, other studies did not show any improvement on total 5 km running time and on cycloergometer incremental test performance in experienced runners and recreationally active men, respectively [31,32]. On the other hand, performance in high intensities or anaerobic sports, requires greater intensity than VO2max and depends on di erent metabolic pathways related to exercise duration [33]. In this sense, while high energetic phosphagen system (<6 s duration) is determinant for explosive disciplines, glycolysis represents the main energetic pathway for exercises between 1–5 min (with an increased contribution of oxidative phosphorylation proportionally with time), determined as high-intensity (~1 min) and intensive e orts (<5 min) [33–36]. Regarding anaerobic performance, chronic Arg supplementation (45–56 days) with low (2 g/day) and high (12 g/day) dosages could led to improve performance in one maximum repetition (1RM) bench press, Wingate test, and VO2max intensity test [9,20]. These positive e ects related with strength could be explained because of Arg enhance GH-releasing hormone, suppresses the endogenous GH-inhibiting hormone and increases insulin-like growth factor 1 (IGF-1) [37,38]. Moreover, Arg plays an essential role in the synthesis of Creatine, main substrate for phosphagen system and anaerobic performance [8]. However, other authors did not nd any bene ts on muscle strength, maximum number of repetitions, and sprint power after ingesting 6 g/day of Arg

GH-releasing hormone, suppresses the endogenous GH-inhibiting hormone and increases insulin-like growth factor 1 (IGF-1) [37,38]. Moreover, Arg plays an essential role in the synthesis of Creatine, main substrate for phosphagen system and anaerobic performance [8]. However, other authors did not nd any bene ts on muscle strength, maximum number of repetitions, and sprint power after ingesting 6 g/day of Arg in both acute and chronic protocols [39–41]. Although Arg supplementation could be e ective on aerobic and high-intensity sport disciplines performance mediated by several e ects, current evidence is controversial and confusing. In this sense, there are a few systematic reviews that analyze the e ects of Arg supplementation on di erent physiological and metabolic mechanisms in older adults and disease patients [42,43]. Moreover, a short

Nutrients2020,12, 1300 3 of 20 systematic review examined the connection between Arg and citrulline and sport performance [15]. However, to the best of the authors' knowledge, there is not clearly and quantitatively analyzed information and distinction regarding the di erent involved e ects in both aerobic and anaerobic performance capacities in the literature, and this may be necessary to better understand Arg supplementation reasons and protocols. Therefore, we proposed carrying out a systematic review and meta-analysis on the e ects of Arg supplementation on exercise performance according to the main energy metabolism system used during exercise, with the main aim of analyzing current evidence and evaluating its impact on performance in both, aerobic and high-intensity or anaerobic, disciplines. In addition, this manuscript aims to show the e ective doses and ideal moment of its intake. 2. Methods 2.1. Literature Search Strategies This systematic review and meta-analysis was performed in accordance with PRISMA ® (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement guidelines [44] and the PICOS model for the de nition of the inclusion criteria: P (Population): “athletes”, I (Intervention): “impact of the Arg supplementation on sport performance”, C (Comparators): “same conditions with control or placebo”, O (Outcome): “sport performance”, and S (study design): “clinical trial” [45]. A systematic search of the scienti c literature was performed to investigate the e ect of Arg supplementation on sports performance. Studies were found by searching Web of Science (WOS), PubMed/MEDLINE, and Scopus from inception to 23rd January 2020, using the following Boolean search equation: “L-Arginine”[All Fields] OR “arginine”[All Fields] OR “AAKG”[All Fields] OR “arginine alpha-ketoglutarate”[All Fields] AND supplementation[All Fields] AND ((“sports”[MeSH Terms] OR “sports”[All Fields] OR “sport”[All Fields]) OR (“exercise”[MeSH Terms] OR “exercise”[All Fields])) AND (“endurance”[All Fields] OR “performance”[All Fields] OR “aerobic”[All Fields] OR “anaerobic”[All Fields] OR “strength”[All Fields]). Over this search equation, other articles in this eld were included by the snowball strategy. To identify duplicates and any potential missing studies, all titles and abstracts from the search were cross-referenced. The abstracts and titles were screened for a subsequent full-text review. 2.2. Inclusion and Exclusion Criteria The inclusion criteria applied

“aerobic”[All Fields] OR “anaerobic”[All Fields] OR “strength”[All Fields]). Over this search equation, other articles in this eld were included by the snowball strategy. To identify duplicates and any potential missing studies, all titles and abstracts from the search were cross-referenced. The abstracts and titles were screened for a subsequent full-text review. 2.2. Inclusion and Exclusion Criteria The inclusion criteria applied in this systematic review and meta-analysis to choice studies were (1) well-designed experiments that included Arg supplementation; (2) identical experimental condition in the placebo or control group; (3) testing the e ects of Arg supplementation on sports performance; (4) clinical trial; (5) clear information concerning the Arg supplementation administration (timing and dosage); (6) published in any language; (7) clear information about funding sources; and (8) absence of con ict of interests. On the other hand, this exclusion criteria were used regarding the experimental procedures of the investigation: (1) Arg supplementation was mixed with other supplements or was a multi-ingredient compound; and (2) participants had a previous injury or health problems. There were no lters applied to the athletes' athletic level, age, gender, or ethnicity to increase the analytic power of the analysis. 2.3. Study Selection Two authors independently screened and agreed upon the selected studies for eligibility (A.V. and J.M.-A.). Likewise, after the inclusion/exclusion criteria were applied to each study, data on study source (including authors and year of publication), sample size, characteristics of the participants (level, race, and gender), supplement administration (dose and timing), and nal outcomes of the interventions were extracted independently by two same authors (A.V and J.M.-A.) using a spreadsheet (Microsoft Inc, Seattle, WA, USA). Then, possible disagreements were resolved through discussion until a consensus was reached, or by third-party adjudication (J.F.-L.). In this sense, the Cohen's kappa

Nutrients2020,12, 1300 4 of 20 coe cient, which indicate the interrater reliability, between authors was above 90 with a level of agreement “almost perfect” [46]. 2.4. Outcome Measures The literature was examined regarding the e ects of Arg supplementation on sports performance using athletic performance outcome variables classi ed according to the duration of tests used. As 5 min test seems to be reliable in determining maximal aerobic velocity and therefore, how long a subject can maintain the lowest intensity at which VO2max was achieved [47,48], authors established this criteria to arrange analyzed performance outcomes variables in VO2max and>VO2max. Two studies met both criteria and, hence, were included in both performance analysis. Concretely, the outcomes obtained by Repeated Sprint Ability Test (RSAT), strength exercises as isokinetic exion, isokinetic extension, and bench press, 1 min all out test, 1 km time trial (TT) and Wingate Test were included in>VO2max. On the other hand, the outcomes obtained by Incremental test to exhaustion, 2 5 km TT, 60 min test at 80% of ventilatory threshold (VT), a 16.1 km TT and 2 6 min running followed by a running test until exhaustion. Harvard Step Test to measure VO2max. capacity were included in VO2max. For the statistical analysis, the sample sizes, means and standard deviations of the di erent outcomes studied were extracted both in the group supplemented with Arg and in the control group and in the pre and post treatment. When there was no numerical data, it was requested to the authors or if the data were plotted as gures, the values were estimated based on the pixel count using images calibrated in ImageJ software (National Institutes of Health, Bethesda, MD, USA). 2.5. Publication Bias Publication bias was assessed using Egger's statistic test, where bias was deemed to be present atp= <0.05 [49]. Corresponding funnel plots were created for visual interpretation, followed by an Egger's statistic to con rm or refute publication bias (Figure). Egger's analyses suggest that publication bias did not present nding in anaerobic performance (z=0.786;p=0.432). However, funnel plot showed publication bias in aerobic performance data (z=2.873;p<0.05).Nutrients 2020, 12, x

test, where bias was deemed to be present atp= <0.05 [49]. Corresponding funnel plots were created for visual interpretation, followed by an Egger's statistic to con rm or refute publication bias (Figure). Egger's analyses suggest that publication bias did not present nding in anaerobic performance (z=0.786;p=0.432). However, funnel plot showed publication bias in aerobic performance data (z=2.873;p<0.05).Nutrients 2020, 12, x FOR PEER REVIEW 4 of 22 2.4. Outcome Measures The literature was examined regarding the effects of Arg supplementation on sports performance using athletic performance outcome variables classified according to the duration of tests used. As 5 min test seems to be reliable in determining maximal aerobic velocity and therefore, how long a subject can maintain the lowest intensity at which VO 2max was achieved [47,48], authors established this criteria to arrange analyzed performance outcomes variables in ≤VO 2max and >VO 2max. Two studies met both criteria and, hence, were included in both performance analysis. Concretely, the outcomes obtained by Repeated Sprint Ability Test (RSAT), strength exercises as isokinetic flexion, isokinetic extension, and bench press, 1 min all out test, 1 km time trial (TT) and Wingate Test were included in >VO 2max. On the other hand, the outcomes obtained by Incremental test to exhaustion, 2 × 5 km TT, 60 min test at 80% of ventilatory threshold (VT), a 16.1 km TT and 2 x6 min running followed by a running test until exhaustion. Harvard Step Test to measure VO 2 max. capacity were included in ≤VO 2 max. For the statistical analysis, the sample sizes, means and standard deviations of the different outcomes studied were extracted both in the group supplemented with Arg and in the control group and in the pre and post treatment. When there was no numerical data, it was requested to the authors or if the data were plotted as figures, the values were estimated based on the pixel count using images calibrated in ImageJ software (National Institutes of Health, Bethesda, MD, USA). 2.5. Publication Bias Publication bias was assessed using Egger’s statistic test, where bias was deemed to be present at p =

no numerical data, it was requested to the authors or if the data were plotted as figures, the values were estimated based on the pixel count using images calibrated in ImageJ software (National Institutes of Health, Bethesda, MD, USA). 2.5. Publication Bias Publication bias was assessed using Egger’s statistic test, where bias was deemed to be present at p = < 0.05 [49]. Corresponding funnel plots were created for visual interpretation, followed by an Egger’s statistic to confirm or refute publication bias (Figure 1). Egger’s analyses suggest that publication bias did not present finding in anaerobic performance (z = 0.786; p = 0.432). However, funnel plot showed publication bias in aerobic performance data (z = 2.873; p <0.05). (a) ( b) Figure 1. Funnel plot of standard error of anaerobic (a) and aerobic; (b) performance data by Hedges’ g. SE: standard error; SMD: standardized mean difference. 2.6. Quality Assessment of the Experiments In accordance with the Cochrane Collaboration Guidelines [50], methodological quality and risk of bias were evaluated by two authors independently (A.V. and J.M.-A.), and disagreements were resolved by discussion and/or third-party author (J.F.-L.). The interrater reliability (Cohen´s kappa) was 90 with a level of agreement “almost perfect” [46]. The list was separated in six different domains: selection bias (random sequence generation, allocation concealment); performance bias (blinding of participants and researchers); detection bias (blinding of outcome assessment); attrition bias (incomplete outcome data); reporting bias (selective reporting); and other types of bias. The domains were considered as ‘low’ if criteria met a low risk of bias (probable bias unlikely to seriously alter the results) or ‘high’ if criteria presented a high risk of bias (probable bias that seriously weakens Figure 1. Funnel plot of standard error of anaerobic (a) and aerobic; (b) performance data by Hedges' g. SE: standard error; SMD: standardized mean di erence. 2.6. Quality Assessment of the Experiments In accordance with the Cochrane Collaboration Guidelines [50], methodological quality and risk of bias were evaluated by two authors independently (A.V. and J.M.-A.), and disagreements were resolved by discussion and/or third-party author (J.F.-L.). The interrater reliability (Cohen's kappa) was

aerobic; (b) performance data by Hedges' g. SE: standard error; SMD: standardized mean di erence. 2.6. Quality Assessment of the Experiments In accordance with the Cochrane Collaboration Guidelines [50], methodological quality and risk of bias were evaluated by two authors independently (A.V. and J.M.-A.), and disagreements were resolved by discussion and/or third-party author (J.F.-L.). The interrater reliability (Cohen's kappa) was 90 with a level of agreement “almost perfect” [46]. The list was separated in six di erent domains: selection bias (random sequence generation, allocation concealment); performance bias (blinding of participants and researchers); detection bias (blinding of outcome assessment); attrition bias (incomplete outcome data); reporting bias (selective reporting); and other types of bias. The domains were considered as

Nutrients2020,12, 1300 5 of 20 `low' if criteria met a low risk of bias (probable bias unlikely to seriously alter the results) or `high' if criteria presented a high risk of bias (probable bias that seriously weakens con dence in the results), or it was considered `unclear' (plausible bias that raises some doubt about the results), if the risk of bias was unknown. Full details or each article and domains are presented in Figures.Nutrients 2020, 12, x FOR PEER REVIEW 5 of 22 confidence in the results), or it was considered ‘unclear’ (plausible bias that raises some doubt about the results), if the risk of bias was unknown. Full details or each article and domains are presented in Figures 2 and 3. Random Sequence Generation (Selection Bias) Allocation Concealment (Selection Bias) Blinding of Participants and Personnel (Performance Bias) Blinding of Outcome Assessment (Detection Bias) Incomplete Outcome Data (Attrition Bias) Selective Reporting (Reporting Bias) Other Bias Abel et al., 2005 Alvares et al., 2012 Alvares et al., 2014 Bailey et al., 2015 Birol et al., 2019 Camic et al., 2010 Campbell et al., 2006 Da silva et al., 2014 Forbes et al., 2013 Greer & Jones, 2011 Hurst et al., 2014 Liu et al., 2009 Meirelles & Matsuura, 2018 Mor et al., 2018 Olek et al., 2010 Pahlavani et al., 2017 Vanhatalo et al., 2013 Yavuz et al., 2014 Figure 2. Summary of risk of bias: authors’ judgements about each risk of bias item for all included studies. indicate low risk of bias; indicate unknown risk of bias; indicate high risk of bias. Figure 2. Summary of risk of bias: authors' judgements about each risk of bias item for all included studies.Nutrients 2020, 12, x FOR PEER REVIEW 5 of 22 confidence in the results), or it was considered ‘unclear’ (plausible bias that raises some doubt about the results), if the risk of bias was unknown. Full details or each article and domains are presented in Figures 2 and 3. Random Sequence Generation (Selection Bias) Allocation Concealment (Selection Bias) Blinding of Participants and Personnel (Performance Bias) Blinding of Outcome Assessment (Detection

This study reviews the impact of arginine supplementation on athletic performance.