Effects of Citrulline Supplementation on Different Aerobic Exercise Performance Outcomes: A Systematic Review and Meta-Analysis

ion with Citrulline (Cit) has been shown to have a positive impact on aerobic exercise performance and related outcomes such as lactate, oxygen uptake (VO 2) kinetics, and the rate of perceived exertion (RPE), probably due to its relationship to endogenous nitric oxide production. However, current research has shown this to be controversial. The main objective of this systematic review and meta-analysis was to analyze and assess the effects of Cit supplementation on aerobic exercise performance and related outcomes, as well as to show the most suitable

and the rate of perceived exertion (RPE), probably due to its relationship to endogenous nitric oxide production. However, current research has shown this to be controversial. The main objective of this systematic review and meta-analysis was to analyze and assess the effects of Cit supplementation on aerobic exercise performance and related outcomes, as well as to show the most suitable doses and timing of ingestion. A structured literature search was carried out by the PRISMA ® (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and PICOS guidelines in the following databases: Pubmed/Medline, Scopus, and Web of Science (WOS). A total of 10 studies were included in the analysis, all of which exclusively compared the effects of Cit supplementation with those of a placebo group on aerobic performance, lactate, VO 2, and the RPE. Those articles that used other supplements and measured other outcomes were excluded. The meta-analysis was carried out using Hedges' g random effects model and pooled standardized mean differences (SMD). The results showed no positive effects of Cit supplementation on aerobic performance (pooled SMD = 0.15; 95% CI ( 0.02 to 0.32);I 2 , 0%;p= 0.08), the RPE (pooled SMD = 0.03; 95% CI ( 0.43 to 0.38);I 2 , 49%;p= 0.9), VO 2 kinetics (pooled SMD = 0.01; 95% CI ( 0.16 to 0.17);I 2 , 0%;p= 0.94), and lactate (pooled SMD = 0.25; 95% CI ( 0.10 to 0.59);I 2 , 0%;p= 0.16). In conclusion, Cit supplementation did not prove to have any bene ts for aerobic exercise performance and related outcomes. Where chronic protocols seemed to show a positive tendency, more studies in the eld are needed to better understand the effects. Keywords:Citrulline; ergogenic aids; physical performance; nitric oxide; aerobic; endurance 1. Introduction Aerobic or cardiorespiratory endurance exercise places high physiological and metabolic demands on the human body's organic regulatory systems [1]. From a physiological point of view, performance in cardiorespiratory endurance sports, where the effort generally lasts longer than 5 min, depends primarily on three related factors: maximal oxygen uptake (VO2max), which can be de ned as the maximum oxygen-carrying capacity of a subject;

cardiorespiratory endurance exercise places high physiological and metabolic demands on the human body's organic regulatory systems [1]. From a physiological point of view, performance in cardiorespiratory endurance sports, where the effort generally lasts longer than 5 min, depends primarily on three related factors: maximal oxygen uptake (VO2max), which can be de ned as the maximum oxygen-carrying capacity of a subject; the individual lactate threshold (ILT), which has many names and is related to different physiological concepts but which, in short, refers to the intensity of exercise at which the Nutrients2022,14, 3479.

Nutrients2022,14, 3479 2 of 28 concentration of lactate in the blood increases exponentially compared to basal levels due to the inability to be oxidized in the mitochondria; and the ef ciency or running economy, de ned as the oxygen consumption or energy required by an individual to maintain a given running pace [2–6]. In addition, the importance of psychological sensation is of the utmost importance in sport, as it is considered a form of biofeedback integrated into the information and effort regulation system between the central command and the local peripheral mechanisms [7,8]. In competitive sport, a 1–2% improvement in performance can make the difference between winning and losing a competition and is, therefore, a critical difference [9]. To improve sports performance, athletes work with complex, carefully planned training and nutrition systems [10,11]. Nutrition and supplementation are therefore important tools in optimizing performance, especially in endurance and aerobic disciplines [6,12]. In their search for the ultimate performance improvement, athletes often make use of a variety of sports supplements [13]. Among the most-used supplements, proteins and amino acids are the most consumed, with a frequency of use of 35–40% [14]. However, the use of nutritional supplements with functions related to Nitric Oxide (NO) synthase has increased considerably in the eld of sport, due to evidence linking their intake to positive effects on sports performance [12,15,16]. The ergogenic interest in NO-related supplements and their different ways of obtain- ing them focuses on the potential effects that this bioactive compound can have on the body. These include health-enhancing effects such as the regulation of blood ow and blood pressure, the maintenance of gastric integrity, and protection against ischemic tissue damage [17–19]. They have also been found to be related to improved performance in different types of disciplines and through different supplements, improving vasodilation and angiogenesis, causing an increase in mitochondrial respiration and biogenesis, pos- itively impacting glucose uptake, optimizing oxygen regulation, and improving muscle contraction [20–26]. Among the supplements linked to these effects are two non-essential amino acids, namely Arginine (Arg) and Citrulline (Cit) [16,27]. Cit is an amino acid abundantly found in

different types of disciplines and through different supplements, improving vasodilation and angiogenesis, causing an increase in mitochondrial respiration and biogenesis, pos- itively impacting glucose uptake, optimizing oxygen regulation, and improving muscle contraction [20–26]. Among the supplements linked to these effects are two non-essential amino acids, namely Arginine (Arg) and Citrulline (Cit) [16,27]. Cit is an amino acid abundantly found in watermelon (Citrullus vulgaris) and can be synthesized from Arg and another amino acid called Ornithine (Orn), with plasma glutamine and Arg itself being the main precursors [27,28] . Likewise, Cit is a precursor to Arg [29,30]. Therefore, Cit plays an essential role in the bioavailability of Arg and also in the subsequent synthesis of NO [29]. However, unlike Arg, Cit passes directly into the kidneys without being catabolized by the enzyme arginase [31]. Cit itself can inhibit arginase action and limit the catabolism of Arg to Orn [32]. It is therefore hypothesized that dietary Cit intake might be more ef cient in increasing Arg bioavailability and NO synthesis than Arg supplementation itself [28,32]. For this reason, Arg and Cit supplementation has been used in athletes from different disciplines and conditions to improve athletic performance [20,33–37]. A recent systematic review and meta-analysis [24] showed a positive effect of Arg supplementation in different sports disciplines, depending on the energy metabolism used. However, evidence for the effect of Cit on aerobic and cardiorespiratory endurance exercise is inconsistent to date. There are insuf cient studies that have qualitatively and quantitatively analyzed its effect on physical performance and the different variables involved. The physiological demands of endurance sports disciplines are directly related to the ability of the cardiovascular system to provide oxygen and nutrients to the muscles, and the functionality of the peripheral (muscular) structures to obtain the required energy, that is, adenosine triphosphate (ATP), as ef ciently as possible. Therefore, the training, nutrition, and supplementation methods used are of particular importance for athletes in these disciplines. They are therefore of great interest considering the potential bene cial effects that the intake of supplements that exogenously and endogenously stimulate NO synthesis have in relation to

to obtain the required energy, that is, adenosine triphosphate (ATP), as ef ciently as possible. Therefore, the training, nutrition, and supplementation methods used are of particular importance for athletes in these disciplines. They are therefore of great interest considering the potential bene cial effects that the intake of supplements that exogenously and endogenously stimulate NO synthesis have in relation to the demands required by long-duration exercise [20]. The increased synthesis of this bioactive compound could ultimately result in multiple bene cial effects [17,20,38–40].

Nutrients2022,14, 3479 3 of 28 Despite the promising evidence of Cit supplementation being used in both high intensity, power, and strength exercise [41,42], as well as in long-term exercise [43–45], there are no conclusive results about its possible effects. Although acute supplementation with Cit (single or multiple doses within a single day) has been shown to signi cantly increase plasma levels of Arg and NO, no improvement in athletic performance has been observed [46,47]. Similarly, no effect was shown on VO2max, anaerobic threshold, time to exhaustion, and the performance parameters analyzed after doses of Cit of between 6 and 12 gadministered 1–2 h before exercise [43,44,48]. In contrast, supplementation using chronic protocols has shown more promising results in relation to endurance performance variables. For example, 7-day Cit supplementation of 2.4 g/day and 6 g/day of Cit improved performance in a 4 km time trial [49] and VO2kinetics and fatigue tolerance in strenuous exercise on a cycle-ergometer [45], respectively. However, one aspect to consider in the interpretation of these results is the level of the participants in the studies. The best results regarding supplementation with ergogenic aids following the NO route have been mainly related to untrained subjects or to participants who are at low competitive levels, whereas no major differences have been found in athletes with high training levels [20]. With this in mind, this systematic review and meta-analysis aim to qualitatively and quantitatively analyze the impact of Cit supplementation on aerobic performance and related variables such as lactate, VO2kinetics, and the RPE to analyze the current evidence in an orderly and systematic way, to extend the current understanding of the ef cacy, effects, and mechanisms behind sports supplementation. 2. Methods 2.1. Literature Search Strategies This systematic review and meta-analysis were conducted by the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [50] and the PICOS inclusion criteria de nition model: “P” (sample or population): “active”; I (intervention): “impact of Citrulline on sports performance”; C (comparison): “Citrulline supplementation compared to Placebo”; O (outcomes): “aerobic sports performance and related variables”; S (study design): “double-blind, crossover, randomized, clinical trial” [51].

were conducted by the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [50] and the PICOS inclusion criteria de nition model: “P” (sample or population): “active”; I (intervention): “impact of Citrulline on sports performance”; C (comparison): “Citrulline supplementation compared to Placebo”; O (outcomes): “aerobic sports performance and related variables”; S (study design): “double-blind, crossover, randomized, clinical trial” [51]. A review of the scienti c literature was carried out, including those studies that analyzed the effect of Cit supplementation on aerobic sports performance and its related variables during exercise, namely, lactate, the rate of perceived exertion (RPE), and oxygen uptake kinetics (VO2). The search was conducted using the respective search engines of the different scienti c databases, without applying any temporal lter, thus covering all entries from the rst studies on the subject in 2006 until the latest published to date in 2021. Speci cally, the PubMed/MEDLINE, Web of Science (WOS), and Scopus databases were used, using the following Boolean search equation: L-Citrulline [All Fields] OR “Citrulline” [All Fields] OR “watermelon juice” [All Fields] AND supplementation [All Fields] AND ((“sports” [MeSH Terms] OR “sports” [All Fields] OR “sport” [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 “lactate” [All Fields] OR “perceived exertion” [All Fields] OR “oxygen kinetics” [All Fields]). Using this equation, relevant articles in the eld were selected by applying a “snowball” search strategy through the references obtained in the initial search. All titles and abstracts were cross-checked and analyzed to identify duplicates or any potential errors, as shown in the ow chart. To do this, the “duplicate search” option in Mendeley Reference Manager was used. All titles and abstracts were also screened to consider whether reading the full articles would be necessary. The search, screening, and selection of published studies were carried out independently by two authors (A.V.M. and J.M.-A.), according to the proposed inclusion and exclusion criteria.

abstracts were also screened to consider whether reading the full articles would be necessary. The search, screening, and selection of published studies were carried out independently by two authors (A.V.M. and J.M.-A.), according to the proposed inclusion and exclusion criteria.

Nutrients2022,14, 3479 4 of 28 2.2. Inclusion and Exclusion Criteria The inclusion criteria applied in this systematic review and meta-analysis for the selection of the articles to be considered in the study were as follows: - - Studies with an appropriate design, using a double-blind, randomized, crossover methodology. - Studies with 2 well-differentiated groups comparing the effects of Citrulline supple- mentation (experimental group) versus a placebo (control group). - Studies were conducted on active people, recreational athletes, well-trained athletes, and elite or healthy professional athletes, both men and women. - Studies in which the comparison of placebo versus supplementation was only with Citrulline and other possible masking compounds, avoiding the intake of different ergonutritional aids at the same time. - - Studies measuring aerobic physical performance using continuous tests lasting 5 min or more, following the criteria relating to the measurement of VO2max. - 2kinetics. - - The following exclusion criteria were used to discard articles found in the initial search: -Studies in which supplementation did not involve Citrulline alone, including those using a combination of different nitric oxide pathway supplements. - Studies in which there was no real presence of a control group ingesting a substance considered to be a placebo. - performance were used in the interventions, considering the scienti c evidence in the literature, such as Caffeine, Beta-Alanine, Creatine, etc. - Studies in which performance was measured using tests lasting less than 5 min and/or measuring variables related to anaerobic performance. - Finally, no lters were applied concerning the gender, level, or sporting discipline of the subjects participating in the studies, and any research that analyzed the proposed variables in any situation that met the inclusion criteria was considered to be valid. 2.3. Study Selection After searching, the various studies were screened and unilaterally selected for analysis by the author. After applying the inclusion and exclusion criteria, data were extracted from each study as shown in the “Results” section. The source of the studies, including authors, year of publication, and citation information, the sample size and characteristics of the participants (level, age, and gender), the administration of

the various studies were screened and unilaterally selected for analysis by the author. After applying the inclusion and exclusion criteria, data were extracted from each study as shown in the “Results” section. The source of the studies, including authors, year of publication, and citation information, the sample size and characteristics of the participants (level, age, and gender), the administration of the supplement to be analyzed and the placebo to be controlled for (dose, timing), the nal variables to be reviewed (test, intensity, and characteristics), and, nally, the main conclusions of each study were extracted by the author with the help of a spreadsheet (Microsoft Inc., Seattle, WA, USA). Subsequently, the data presented were analyzed and several studies were excluded for relevant reasons until the studies to be included in this systematic review and meta-analysis were obtained. 2.4. Measuring Variables The available scienti c literature on Cit supplementation and its effect on aerobic physical exercise was examined in terms of performance in the physical tests conducted, the analysis of lactate as a determining energy metabolite, the RPE, and VO2kinetics as a physiological factor limiting aerobic performance. To quantify aerobic sports performance, measurement variables classi ed according to the duration of the tests were used. The

Nutrients2022,14, 3479 5 of 28 5 min test appeared to be a reliable test for determining maximal aerobic speed, related to VO2max, and therefore suggested the time that a subject can maintain the lowest intensity related to this physiological requirement [52,53]. Taking into account that VO2 max marks a subject's maximum aerobic power, the author established testslasting 5 min as time criteria for analyzing aerobic sports performance in this systematic review and meta-analysis. More speci cally, several tests were included in the performance analysis to determine aerobic performance, both on the cyclo-ergometer and treadmill, according to the proposed criteria: incremental and sustained tests to exhaustion; Bruce protocol; treadmill test at sub-maximal intensity (40% of HHR); constant work test at 70% of VO2max; moderate intensity test (90% of HHR); constant work test at 70% of VO2max; moderate intensity test (90% of VO2max); moderate intensity test (90% of GET) followed by a high-intensity test on the day (GET + difference between GET and VO2max work); 6 min constant work test (70% VO2max) followed by a 30 s all-out test at 70% of VO2max; a 30 s all-out test; 10 repetitions of 15 s at maximum intensity (sprint); 5 min rest and an incremental test to exhaustion at 100% of individual peak power; a half-marathon race; a 4 km and 40 km individual time trial; and an SRT test comprising a 6 1-min sprint at 120% of maximum power. Likewise, for the analysis of lactate, the measurement of this biomarker was analyzed in the different studies in the same way as for the RPE, using the Modi ed Borg Scale (0–10) or Borg Rating of Perceived Exertion Scale (6–20). In turn, different related variables were used to analyze the responses for VO2kinetics. These included: net oxygen expenditure, mean response time (MRT), oxygen de cit, mean pulmonary VO2response time, mean overall pulmonary gas response time, tissue oxygenation index, muscle oxygenation, pulmonary VO2, VO2max, gas exchange de ned threshold, and peak VO2. For the statistical analysis of the quantitative synthesis (meta-analysis), the sample size, means, and standard deviations of the different variables were extracted for

This systematic review analyzes the effects of Citrulline supplementation on aerobic exercise performance.