Nutritional supplementation is a common strategy used by athletes and recreationally active adults to improve physical performance and muscle recovery. Nitric oxide (NO)-related supplements have received special attention for their possible ergogenic effects [1
]. In particular, citrulline malate (CM) has been reported to augment aerobic energy production during exercise and increase phosphocreatine (PCr) during exercise recovery [2
], improve ammonia (NH3) elimination during recovery from exhaustive exercise [4
], attenuate muscle soreness after high-intensity resistance exercise (RE) [6
], and increase performance during repeated bouts of high-intensity RE [6
CM is formed by combination of L-citrulline (CIT) and malate (or malic acid)—a salt primarily found in apples. The potential ergogenic effects of CM have been attributed to three key mechanisms. First, oral CIT ingestion has been show to increase plasma L-arginine levels [9
] at rest and exercise in humans. Given that L-arginine is the main substrate for synthesis of NO, an important modulator of blood flow [12
], it has been suggested that oral CM supplementation may indirectly increase NO synthesis [6
] and thus increase blood flow to active muscles. In this way, CM supplementation could contribute to increase nutrient delivery and/or clearance of waste products [13
] such as plasma lactate and ammonia, thereby improving muscle function [15
Second, CIT is an essential component of the urea cycle in the liver [16
], where L-arginine produced from CIT is catabolized by arginase into ornithine and urea. Given that urea is the major vehicle to eliminate ammonia, a promoter of muscle fatigue via anaerobic glycolysis and consequent lactic acid production [17
], it has been suggested that CIT supplementation may improve ammonia homeostasis [18
] and thus improving muscle function. Third, malate is an intermediate of the tricarboxylic acid (TCA) cycle, and its greater availability after CM supplementation may augment aerobic ATP production by the TCA cycle through anaplerotic reactions [2
], resulting in decreased muscle fatigue and improved muscle performance [2
Based on the aforementioned mechanisms, it is postulated that CM supplementation may increase muscle performance and recovery by several ways, including reducing muscle soreness [6
] and fatigue [2
], improving oxygen delivery to the muscle [2
], increasing oxidative ATP production during exercise and increasing PCr during exercise recovery [2
], and lowering lactate and ammonium production [19
]. Despite an abundance of studies on ergogenic effects of CM supplementation, no study to date has examined whether CM supplementation improves recovery of muscle function after high-intensity RE in young adult subjects.
The aim of this study was to investigate the effects of free CM supplementation on recovery of muscle function after a single session of high-intensity RE in untrained young adult subjects. Based on the physiological properties and beneficial effects of CM on muscle fatigue and performance [6
], we hypothesized that free CM supplementation would enhance muscle recovery from RE by improving muscle functional, metabolic, anabolic, and physiological responses.
To our knowledge, this is the first study to examine the effects of free CM supplementation on the time course of muscle recovery after a single session of high-intensity RE in untrained young adult men. Based on the physiological properties and beneficial effects of CM on fatigue and performance [2
], we hypothesized that CM supplementation would enhance muscle recovery from RE by improving the muscular functional, metabolic, anabolic, and physiological responses. In contrast to the hypothesis, we observed that free CM supplementation (6 g at 60 min pre-workout) does not improve the major functional (i.e., number of maximum repetitions, muscle soreness, and perceived exertion), metabolic (i.e., CK, and lactate), anabolic (i.e., testosterone:cortisol ratio), and physiological (i.e., RMS and MF signal) indicators of muscle recovery in untrained young adult men.
Given that L-arginine is the main substrate for the NO synthesis, and CIT may be converted to L-arginine in kidney [36
], it has been postulated that oral CM supplementation may increase NO synthesis [6
] and, consequently, improve muscle blood flow for active muscles. As a result, CM supplementation could increase nutrient and oxygen delivery to the muscle and/or enhance clearance of waste-products [13
] such as plasma lactate and ammonia, thereby improving muscle function/recovery from exercise [15
]. Nevertheless, we have shown that CM supplementation does not promote any improvements in markers of muscle function (i.e., number of maximum repetitions, muscle soreness, and perceived exertion) during recovery from RE (24, 48, ad 72 h). In contrast to our results, previous performance studies have shown that CM supplementation (8 g) can reduce rating of perceived exertion [8
] and increase performance (i.e., number of maximum repetitions) during repeated bouts of high-intensity RE to failure [6
]. Although a direct comparison of our findings with the aforementioned studies may not be warranted due to the differences between the studies aims (e.g., recovery vs. performance), an important question is why we did not see improved muscle function during recovery, as others have reported in performance studies [6
A possible explanation may be the inability of CM to attenuate muscle damage during recovery period. The RE session resulted in similar increase in plasma CK (24 h post-exercise) and lactate (immediately post-exercise) levels in both the CM and PL conditions, which typically indicates muscle damage and impaired muscle function. Wax et al. [7
] reported an increase in muscle performance (i.e., number of repetitions performed) without any inhibitory effect of CM on lactate production, indicating that beneficial effects of CM on muscle function are not attributed to lactate reduction. Similarly, Vanuxem et al. [4
] showed no difference in lactate levels between CM and PL groups during a maximal exercise test. Therefore, it seems likely that the lack of effects of CM on muscle function (i.e., number of maximum repetition) observed in our study is more related to inability of CM to attenuate RE-induced muscle damage (indicate by similar CK levels between CM and PL during recovery period) than to reduce lactate production. Considering that muscle damage is a potential indicator of impaired muscle function after intense exercise [37
], its negative impact could be superior to any small beneficial effects of CM supplementation on muscle function during recovery. This could explain, at least partially, why CM supplementation promoted beneficial effects when muscle was tested in normal conditions (without any exercise-induced damage) [6
], but not in our study, where the muscle was exposed to a high degree of muscle damage after RE session.
The absence of a positive effect of CM on muscle regeneration and function during recovery may be due to its particular mechanisms of action. CM has been shown to increase ATP production and phosphocreatine (PCr) resynthesis rate [2
] and improve ammonia (NH3
) elimination during recovery from exhaustive exercise [4
], but CIT does not directly increase muscle protein synthesis rates or the phosphorylation of anabolic signaling proteins (i.e., mammalian target of rapamycin (mTOR)) in human muscles [38
]. This lack of CIT effects on muscular regeneration markers [38
] was supported by the absence of differences in anabolic hormones (i.e., testosterone:cortisol ratio) levels between CM and PL conditions in our study. Therefore, it is likely that the inability to improve anabolic factors result in no beneficial effect of CM supplementation on muscle regeneration (i.e., CK levels) and function (i.e., number of maximum repetition) during recovery from RE. This was also evidenced by the observation that muscle soreness did not improve with CM supplementation.
Additionally, we showed no differences in electromyographic (EMG) indicators of muscle activation (RMS) and fatigue (MF) between CM and PL conditions. To our knowledge, this is the first study to examine the effects of CM supplementation on EMG indicators of muscle activation (RMS) and fatigue (MF) during recovery from high-intensity RE. Considering that the decline in MF and increase in RMS are typically associated with muscle fatigue during isometric and dynamic contractions [39
], we expected an inverse effect of CIT supplementation on these factors in the fatigue tests during recovery period. However, no differences were observed between the CM and PL conditions, which is in line with the lack of a beneficial effect of CM supplementation on muscle function (i.e., number of repetitions performed). This indicates that CM supplementation is not effective in improving the neuromuscular responses during recovery from RE. This result is consistent with no difference in the muscle soreness between CM and PL conditions during the recovery period. Although muscle soreness may be a poor indicator of exercise-induced muscle damage during recovery [43
], it usually reflects muscle fatigue. This supports the findings of the present study that CM supplementation does not attenuate muscle fatigue during the course of recovery. Therefore, any possible beneficial effects of CM supplementation on muscle soreness [6
], fatigue [2
], oxygen delivery to the muscle [2
], aerobic energy production [2
], and lactate and ammonium clearance [18
] may not be sufficient to improve muscle recovery in untrained young adult men. It is noteworthy that a dose of 6 g of CM is sufficient to increase plasma CIT (~173% increase) and arginine (~123% increase) levels [10
], and NO production after exercise (measured as nitrite levels) [10
]. In addition, is has been demonstrated that after administration of CIT (5–10 g), time to reach maximum concentration (Tmax) was ~43 min and decreased to baseline by 3–5 h [44
]. Thus, the lack of a positive effect of CM supplementation on muscle recovery from RE does not necessarily indicate that our subjects did not experience an increase in the plasma CIT and arginine levels during and after exercise.
A few limitations of this study must be mentioned. First, we did not analyze plasma NO and CIT concentrations. However, previous studies that used the same dose of CIT (i.e., 6 g) showed an increase in plasma CIT concentrations [10
]. Second, we did not collect muscle biopsies for analysis of muscle tissue markers of regeneration (e.g., protein synthesis and anabolic factors such as insulin-like growth factor (IGF-I), hepatocyte growth factor (HGF), mTOR, and ribosomal protein S6 kinase (p70S6k)) and damage (e.g., histological changes); however, we analyzed the major plasma markers of muscle damage and regeneration (i.e., CK and testosterone:cortisol ratio) as well as functional outcome measures (i.e., number of repetitions performed, perceived exertion, muscle soreness, and EMG signs). Finally, we did not standardize the subjects’ diets. However, we did instruct subjects to duplicate their food intake for the 24 h proceeding each session.
In conclusion, CM supplementation (6 g dose at 60 min pre-workout) does not improve muscle recovery from a single session of high-intensity RE in untrained young adult men. Therefore, it is premature to recommend free CM supplementation as an ergogenic aid to improve muscle recovery after RE. Future studies are required to assess the effects of CM in other populations (e.g., elderly and/or women) with different training status (e.g., recreational practitioners and/or athletes).