The Effect of Citrulline Malate Supplementation on Athletic Anaerobic Performance

B. R. J. ADAMS

Loughborough College, Radmoor Road, Loughborough UK, LE11 3BT

Submitted 20 March 2013

The aim of this study was to examine the ergogenic effects of CM supplementation on athletic anaerobic performance. Eight male subjects (age 21.34 ± 0.51 years, body mass 78.98 ± 11.60 kg, height 178.25 ± 6.54 cm) completed a reps-to-fatigue test; following a series of bench press exercises (3 exercises x 3 sets; with 1 minute rest periods between sets, 2 minutes between exercises) on 3 separate occasions over a 4-week period. A familiarisation test was included, where 1RM’s were established. On the other 3 occasions subjects ingested CM (4g or 8g) or a placebo in a randomised double blind crossover design. All trials were conducted using the same equipment in the same fitness gym. There was no significant difference (P >0.05) in the amount of repetitions performed between exercise1 and exercise1b (4g CM, 8g CM vs placebo: 16 ± 1.90, 17.0 ± 2.16 vs 16.46 ± 2.60). There was no significant difference in the amount of muscle soreness 24 hours (4g CM, 8g CM vs placebo: 2.86 ± 2.03, 0.24 ± 0.46 vs 1.68 ± 1.30) and 48 hours after exercise (4g CM vs placebo: 1.56 ± 1.20, vs 0.66 ± 0.66) between CM and placebo supplemented trials (no reports of muscle soreness were recorded for 48hrs with 8g CM). In addition, no recordings of gastrointestinal discomfort were experienced. These results suggest that CM supplementation has no effect on anaerobic performance or the amount of muscle soreness experienced post exercise.

Keywords: anaerobic exercise, ergogenic aids, sport performance, weight training

Introduction

Journal of Sport Sciences, 2013, 01, 001-028

1

Citrulline malate (CM) is a nutritional supplement that has been recently researched as a potential ergogenic aid towards both aerobic and anaerobic performance (Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002). Despite its current popularity as a sports supplement, CM has only been authorised as a pharmaceutical drug for Asthenia (Creff, 1982; Pérez-Guisado and Jakeman, 2010), however, it is believed to have a significant effect upon higher performance and faster recovery following intensive training when used as an ergogenic aid with dosages between 4-10 grams (g), one hour before exercise (Pérez-Guisado and Jakeman, 2010). The exact mechanisms of CM remain elusive in regards to its effect upon sports performance (Pérez-Guisado and Jakeman, 2010; Bendahan et al., 2002), however there are several hypothetical considerations for its biochemical breakdown.

L-citrulline has an important role in the urea cycle accelerating the removal of ammonia (Figure 1), which is a toxic by-product of muscle protein breakdown that can inhibit aerobic utilisation of pyruvate if accumulated (Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010). This accumulation of ammonia in the blood and muscle tissues may have an impact on muscular fatigue due to the blockage of pyruvate utilisation causing a lactic acid by-product hindering performance (Vanuxem et al., 1986). The lack of oxygen during the oxidative pathway determines the fate of pyruvate and its definitive completion as either lactate or energy (Wilmore, Costill and Larry Kenney, 2008). Therefore, without the presence of oxygen, the oxidative pathway produces lactate as a by-product. Additionally, the presence of ammonium with heavy exercise causes a metabolic block, decreasing glycolytic production, thus obstructing performance (Insel et al., 2012). Ammonium is toxic to the body and, if possible, should be avoided throughout energy production (Insel et al., 2012).

Malate is an intermediate in the tricarboxylic acid cycle (TCA) and has a beneficial effect on energy metabolism by facilitating aerobic ATP production (Gropper and Smith, 2012; Pérez-Guisado and Jakeman, 2010). Primarily, it has been shown to bypass the blockage caused by ammonia in the oxidative pathway, limiting the accumulation of lactic acid, which causes a re-direction towards pyruvate genesis (Pérez-Guisado and Jakeman, 2010). Thus, aerobic energy metabolism can take place (Nelson and Cox, 2005).

L-citrulline is a non-essential amino acid naturally occurring in the body. Its name is derived from the Latin word for watermelon, citrullus, from which it was first isolated in a study by Wada (1930). More recently, Rimando and Perkins-Veazie (2005) have researched its content in watermelon rind, providing evidence to support watermelon as the main dietary source. L-citrulline is created endogenously within two main pathways (Béscos et al., 2012). 1) Through ornithine and carbamoyl phosphate synthesis via arginine and (Figure 1. The metabolic pathway of L-citrulline through the urea cycle. Image by (Lehninger, 1982))glutamine condensation in enterocytes during a reaction catalysed by ornithine carbamyl-transferase (Kaore, Amane and Kaore, 2013; Endo et al., 2004). 2) As a by-product during the conversion of L-arginine into Nitric Oxide (NO) in a reaction catalysed by Nitric Oxide Synthase (NOS) enzymes (Béscos et al., 2012; Sureda et al., 2010).

Recently, dietary interest has increased due to the importance of L-citrulline as a precursor of L-arginine (Béscos et al., 2012). L-citrulline bypasses hepatic metabolism and is taken up by the liver and other tissues to be converted into L-arginine (Hartman, Torre and Prior, 1994), whereas L-arginine is degraded in the liver, thus only small amounts can be utilised (van de Poll et al., 2007; Hickner et al., 2006). It has been suggested that systemic administration of L-citrulline may be a way of increasing extracellular levels of L-arginine as opposed to L-arginine ingestion alone (Hartman, Torre and Prior, 1994). Additionally, L-citrulline has been shown to increase L-arginine plasma concentration in sickle cell patients by 60% and to almost double the concentration in healthy humans (Hickner et al., 2006). Further research shows a 30% increase in L-arginine levels with L-citrulline supplementation (3-6g) (Waugh et al., 2001). Therefore, L-citrulline may prove as a sufficient means for elevating L-arginine levels indirectly, hence aiding sports performance through the production of NO.

NO production can be generated through the absorption of green leafy vegetables, such as lettuce, spinach and beetroot, due to their high concentrations of nitrate (80%) (Weitzberg, Heze and Lundberg, 2010), likewise through the breakdown of L-arginine, thus increasing vasodilation, nutrient delivery, glucose uptake and clearance of waste products from the working skeletal muscle (Bean, 2010; Kingwell et al., 2002; Roy, Perreault and Marette, 1998). NO production has been shown to mediate glucose transport into skeletal muscle with the stimulation of exercise (Higaki et al., 2001), which may aid the rate of glycolysis, therefore increasing performance (McConell, 2007).

Vasodilation is caused in the endothelium cells, which can allow for further working capacity due to the expansion of blood vessels (Talbott, 2003). This increase of L-arginine through L-citrulline ingestion could prove beneficial towards anaerobic performance indirectly, increasing plasma levels of L-arginine (Béscos et al., 2012) and modulating blood flow through NO production (Shen et al., 1994).

(Figure 2. The process that malate undertakes during the oxidative pathway (TCA cycle). Image by (Lehninger, 1982))The primary function of NO is its vasodilatory effects; however it is also responsible as a regulatory molecule maintaining physiological functions within the body (Alvares et al., 2011; Pérez-Guisado and Jakeman, 2010). In human studies, L-arginine has been shown to increase substrates responsible for muscle recovery and protein synthesis through an increased production of NO (Alvares et al., 2011), as well as promoting the clearance of ammonium and lactate (Alvares et al., 2011), which are both related to muscular fatigue (Schaefer et al., 2002). It is thought that the efficiency of the recycling systems could have an effect on the production of NO in relation to L-citrulline production at a ratio of 8:1 (Flam, Eichler and Solomonson, 2007).

Malate, an intermediate of the Tricarboxylic acid cycle (TCA) is able to act as a shuttle between cytoplasm and mitochondria, bypassing a blockage caused in the oxidative pathway induced by ammonia (Pérez-Guisado and Jakeman, 2010). Malate is freely permeable and is oxidised by malate dehydrogenase into oxaloacetic acid in the matrix of the mitochondria (Figure 2) (Hames and Hooper, 2000). It plays an important role in the production of Adenosine Triphosphate (ATP) because it produces NADH (nicotinamide adenine dinucleotide), which enters the electron transport chain and creates 2.5 ATP’s per mole (Gropper and Smith, 2012; Hames and Hooper, 2000). During aerobic glycolysis when total oxygen consumption is sufficient to allow for glucose utilisation, lactate is not formed (Gropper and Smith, 2012). Instead, pyruvate is used and enters the mitochondrion and NAD+ is reduced (Hames and Hooper, 2000). In order for ATP to be generated, NADH must be transferred or shuttled into the mitochondria though a mechanism known as the malate-aspartate shuttle system (Gropper and Smith, 2012; Wu et al., 2007). This process is believed to enhance energy production (Wagenmakers, 1998) and could therefore benefit the current study through a faster rate of ATP production.

Studies have also shown that malate is an important trigger for oxidation of acetyl-CoA in β-oxidation of fatty acids, aiding the formation of ATP (Wu et al., 2008). An earlier study by Wu et al. (2007) suggested that malate supplementation might improve physical endurance whilst enhancing the activity of the malate-aspartate shuttle. However, this study was performed on mice and therefore may not have the same effects on human performance (Hickner et al., 2006; Meneguello et al., 2003).

There are several studies that report the possible effects of CM on aerobic and anaerobic performance in both human and animal studies (Giannesini et al., 2011; Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Giannesini et al., 2009; Bendahan et al., 2002). However, there is very little evidence on the individual components that have been combined to form CM. One study has researched the effects of L-citrulline supplementation alone, on sports performance (Hickner et al., 2006). This study was administered through an incremental time to exhaustion treadmill test. Healthy young subjects took dosages of 3g (3 hours) and 9g (24 hours) of L-citrulline before the test. Results suggest that supplementing with L-citrulline itself impairs time to exhaustion exercise performance compared with a placebo. However, studies provide controversial evidence, showing an increased time to exhaustion following L-citrulline supplementation in rats (Meneguello et al., 2003) but a decrease in humans (Hickner et al., 2006). The authors linked this response to the proposed effect of L-citrulline on insulin secretion (Wascher et al., 1997) and glucose uptake (McConell et al., 2005) due to increased levels of NO.

Whilst evidence provided by Hickner et al. (2006) suggests that L-citrulline potentially has no effect on exercise performance, there is extremely little evidence to support the effect of its corresponding substance in human studies (Wu et al., 2008; Wu et al., 2007). Nonetheless, research has been conducted to support the use of CM as a pharmaceutical treatment (Creff, 1982) and its hypothetical use as an ergogenic aid (Giannesini et al., 2011; Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002). Currently, CM is used as a treatment for people suffering with asthenia (under the brand name Stimol) 1g 3 times daily (Pérez-Guisado and Jakeman, 2010). Creff (1982) reported the effects of CM on people complaining of fatigue and found a significant effect with dosages of 4g/day (2g before each of 2 main meals). The safety/acceptability of the supplement was also deemed very significant with minor manifestations of intolerance of a digestive nature (Creff, 1982). This could indicate that a lower dosage may be required to eliminate feelings of gastrointestinal discomfort experienced in a previous study (Pérez-Guisado and Jakeman, 2010).

More recently, studies have been carried out exploring the influence of CM on aerobic and anaerobic performance (Giannesini et al., 2011; Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002). Giannesini et al. (2011) investigated the effects of oral administration of CM on skeletal muscle function in healthy rats. Findings suggest that supplementation of CM enhances muscle force production at a decreased energy cost of contraction. However, due to the examination of the study, there is evidence to suggest that this may differ slightly from human trials (Hickner et al., 2006; Meneguello et al., 2003). Dosages in the preceding study were 3g/kg/day, which under the current dosages advocated for the treatment of asthenia (1g 3 times daily) (Creff, 1982) and as an ergogenic aid (4-10g, one hour before exercise (Pérez-Guisado and Jakeman, 2010), it might seem a substantially high amount if it were to be conveyed in human studies. Therefore, further research is needed in determining the required dosages for use of CM as an ergogenic aid for sporting performance (Béscos et al., 2012)

In human studies, CM dosages are inconclusive with three studies (Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002) advocating varying quantities for increased performance. For example, Bendahan et al. (2002) examined the effects of CM on finger flexions in 18 men complaining of fatigue. Ingestion resulted in a 34% increase of ATP production, and a 20% increase in the rate of phosphocreatine recovery after exercise, following oral supplementation of CM, 6g/day for 15 days. This increase in the rate of oxidative ATP production supports evidence based on the theoretical effects of malate, its role in the TCA cycle, and its documented facilitation of ATP production (Hames and Hooper, 2000). Further research also supports the increased rate of oxidative ATP produced (Wu et al., 2008; Wu et al., 2007). However, caution should be taken under the examination of these results due to the simplicity of the design, the nature of the test, and the criticism in regards to the reliability of the investigation (Béscos et al., 2012). For example, testing conditions were performed without a blind condition or a placebo, additionally; finger flexions have little to no relation to sports performance, therefore, supplying little significance for its effect on aerobic energy production (Béscos et al., 2012; Pérez-Guisado and Jakeman, 2010; Bendahan et al., 2002).

More recently, Sureda et al. (2010) investigated the aerobic effects of CM on exercise performance during a 137-km cycling stage on branched chain amino acid (BCAA) utilisation. Nevertheless, despite its differences there are some valid conclusions drawn. Participants ingested 6g of CM supplementation, 2 hours before the test, which resulted in an increase in growth hormone and the utilisation of BCAA’s during exercise. This increase in growth hormone could be largely beneficial, especially for strength performance and/or hypertrophic gains (Lemon, 1991). Increased levels of BCAA’s could prove as a beneficial finding, however, further research is needed (Williams, 2005). Correspondingly, a study by Pérez-Guisado and Jakeman (2010) reported the effects of CM supplementation on anaerobic performance (resistance training) and muscle soreness over a 3-week period. The study indicates that CM supplementation may confer a training or performance benefit, with athletes that participate in high intensity anaerobic exercise. 41 healthy men performed a reps-to-fatigue test with measurements taken from 8 sets of the barbell bench press (total of 16 sets, measurements taken from first 4 and last 4 sets) at 80% 1RM. The test was performed in a randomised, double-blind manner with a placebo and an 8g CM solution, one hour before testing. However, more recently, published literature shows that after 10 weeks, 3-times/week resistance training with differing loads (light 30% 1RM and high 80% 1RM) in unilateral knee extension, hypertrophy gains can be found in both lower and higher loads to failure (Mitchell et al., 2012). Thus, providing evidence to suggest that loads advocated by Pérez-Guisado and Jakeman (2010), may be unnecessary for required results.

Pérez-Guisado and Jakeman (2010) suggests findings of gastrointestinal discomfort amongst 14.63% of subjects, however, studies have examined the effects of CM on sports performance and in conclusion reported no findings in regards to gastrointestinal discomfort (Sureda et al., 2010; Bendahan et al., 2002). Nevertheless, there was no protocol in place to examine the effects of CM on gastrointestinal discomfort in these studies (Sureda et al., 2010; Bendahan et al., 2002); therefore the significance of their findings could be considered incomplete. Pérez-Guisado and Jakeman (2010) supplied little documentation towards the specific testing protocols used for gastrointestinal discomfort, hence the current need for an established, valid and reliable scale for its measurement.

Recent literature supplies supporting evidence for the reliability of a visual-analogue scale (VAS). Markers on a scale of 1 to 15 are used for the measurement of gastrointestinal discomfort experienced (Siegler et al., 2012). Similarly, the current method used by Pérez-Guisado and Jakeman (2010) for the reporting’s of muscle soreness post-exercise is of limited agreement (Béscos et al., 2012; Flann et al. 2011). Current studies support the proposed use of the VAS similarly to the gastrointestinal discomfort recommendations (Hawker et al., 2011; O'Connor et al., 2000). Flann et al. (2011) used this particular scale in conjunction with further advanced methods including creatine kinase levels and muscle biopsy for testing levels of muscle soreness. They found that the VAS provided reliable results when compared with creatine kinase and muscle biopsy techniques.

The current study examines the effect of CM on athletic anaerobic performance through a bench press protocol presented by Pérez-Guisado and Jakeman (2010). However, the contents of their experiment have been altered slightly, based on the recent findings supported by Mitchell et al. (2012). Due to its safety and acceptability as a drug, widely used for the treatment of asthenia (Creff, 1982); its potential enhancement on sports performance under both aerobic and anaerobic conditions (Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002); and evidence to support its fatigue-resistant properties (Giannesini et al., 2009) after resistance exercise performance (Pérez-Guisado and Jakeman, 2010). It is suggested that CM could have a significant effect on anaerobic performance. However, due to the nature of the current research suggesting a potential impact upon gastrointestinal discomfort (Pérez-Guisado and Jakeman, 2010), it could be contested that 8g of CM supplementation is a considerable amount for human performance (Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002). Thus, postulating evidence to suggest that current dosages remain inconclusive.

It is hypothesised that CM may have an impact on anaerobic performance, increasing repetitions and decreasing muscle soreness over a 48 hour period. It is further assumed that gastrointestinal discomfort may be experienced, following the ingestion of 8g CM, as reported by Pérez-Guisado and Jakeman, (2010). Therefore, the aim of the current study was to examine the effect of 4 and 8g of CM supplementation on the total amount of repetitions performed during a bench press protocol; the effect that CM has upon gastrointestinal discomfort; and the total amount of muscle soreness experienced 24 and 48 hours following each session.

Methodology

Participants

Eight physically active (>six months strength-training experience, 4 ± 2 workouts per week, body mass 78.98 ± 11.60 kg) healthy young (age 21.34 ± 0.51 years, height 178.25 ± 6.54 cm) male subjects were studied during a reps-to-fatigue test. They were recruited from Loughborough University and Loughborough College, Leicestershire.

To be included in the study participants were asked to sign an agreement that would require attendance to the gym (>3 times/week) following a guided training plan; refrain from sporting activity during the weeks of testing; must not have taken anabolic steroids present or past; must not have ingested HMB, creatine, thermogenics, or any form of ergogenic aid that may affect performance for an 8-week period; not have taken or been taking non-prescription drugs or nutritional supplements during the test; must not have any existing medical conditions that could affect their participation and must stick to their usual diet. Participants were asked to record a 48-hour food diary (Bingham et al., 1997) prior to the test and were asked to follow the same habitual diet for each following test with the avoidance of foods containing high levels of nitrate (green leafy vegetables) (Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Hickner et al., 2006). Written informed consent was obtained from each participant along with a Par-Q health-screening questionnaire. Loughborough College ethics committee approved the study.

Experimental Approach

Participants completed a reps-to-fatigue test following a similar protocol established by Pérez-Guisado and Jakeman (2010). The study was conducted at Loughborough College using a randomised, double-blind crossover design. A warm-up consisting of 10 minutes cycling on an exercise bike (Life Fitness 9500HR, Cambridgeshire, UK) in accordance to their 60% age-related heart rate (Chandler and Brown, 2008), followed by 15-20 reps dynamic movement with a comfortable weight (LeSuer et al., 1997) (Eleiko Sport AB, Korsvägen, Sweden) chosen by the participant, before each test. Subjects attended a total of three sessions (4g CM, placebo, and 8g CM) and a familiarisation test where their one repetition maximum (1RM) was determined. The reps-to-fatigue test consisted of three exercises; flat barbell bench press (exercise1) (Eleiko Sport AB, Korsvägen, Sweden), incline barbell bench press (exercise2) (Eleiko Sport AB, Korsvägen, Sweden), and flat barbell bench press (exercise1b) (Eleiko Sport AB, Korsvägen, Sweden) with a total of 9 sets. Exercise1 was performed at 60% of their predetermined 1RM for 3 sets, followed by exercise2 at 50% predetermined 1RM for 3 sets, and finally exercise1b at 60% predetermined 1RM for 3 sets. Rest intervals of 1 minute between sets and 2 minutes between exercises were instructed and recorded using a stopwatch (Sport Timer 220, Sportline, New York) (Pérez-Guisado and Jakeman, 2010), whilst all subjects were required to perform each set to muscular failure following a 2-2 tempo (Pérez-Guisado and Jakeman, 2010). A total of eighteen measurements were taken for each subject (6 for the 4g CM, 6 for the placebo and 6 for the 8g CM) along with recordings of muscle soreness (measured in mm’s), 24 and 48-hours after testing using a recognised VAS (Flann et al., 2011; Hawker et al., 2011).

Subjects were randomly assigned to consume beverages one hour before testing (Pérez-Guisado and Jakeman, 2010) consisting of 4g and 8g of CM (Myprotein, England), 20 mL lemon juice, 10g powdered sugar, 40 mL blackcurrant cordial and tap water to complete a 200 mL solution. A placebo consisting of 40 mL lemon juice, 10g powdered sugar, 40 mL blackcurrant cordial, and tap water to complete a 200 mL solution was also provided (beverages were shaken and provided in solid colour water bottles). The investigator who made the solutions took no part in performing the bench press protocol and all tests were carried out at the same time/day/place each week allowing for a one-week wash-out period between tests (Pérez-Guisado and Jakeman, 2010). Subjects were told to inform the investigator of any known side effects throughout the test, if so, completion of the VAS (AMDA) (ranging from 1-15) (Siegler et al., 2012) was required to examine gastrointestinal discomfort.

Statistical Analysis

Statistical analyses were run using a statistical software package, IBM® SPSS Statistics (Version 20, Windows 7). Tests for normality were performed using a Shapiro-Wilk for all statistical analyses. Two-way repeated-measures analysis of variance (ANOVA) with Bonferroni adjustments conducted were used to determine the effects of CM supplementation against placebo, on total number of repetitions performed between exercise1 and exercise1b, as well as muscle soreness 24 and 48 hours post-exercise. A one-way ANOVA was run for all significant findings.

Results

CM Supplementation and Placebo

All data was reported as normally distributed. No significant difference was found for the effects of supplement (F (2.0, 14.0)=2.0, P = 0.173) across all three groups. The total number of repetitions performed across all sets was considered significant (F (1.1, 7.8)=57.6, P = <0.001). However, there was no significant difference reported for the interaction between each supplement and the total number of repetitions performed through all sets (F (4.0, 28.0) =2.0, P = 0.116).

(Figure 3. The total number of repetitions performed across a 3-week period. Bars are representative of mean values from exercise1 and exercise1b across all 3 sets. Error bars represent standard deviation.)Muscle Soreness

Data was reported as normally distributed, with the exception of one group (8gCM, 24hrs). A two-way ANOVA reported statistically significant differences for the supplement (F (2.0, 14.0) =10.2, P = 0.002), time (F (1.0, 7.0) =17.2, P = 0.004), and the interaction between supplement and time (F (2.0, 14.0)=6.3, P = 0.011). Therefore, a one-way ANOVA was conducted to determine at which point a significant difference occurs, the findings of the test show there was a significant difference between all three groups (F (2.0, 21.0)=6.9, P = 0.005). A Bonferroni post hoc test revealed that there was no significant difference in decreased muscle soreness between placebo and 4g of CM (P = 0.328); no significant difference between placebo and 8g of CM (P = 0.168); however, there was a significant difference between 4g of CM and 8g of CM (P = 0.004) over 24 hour periods. A one-way ANOVA presented that there was a statistically significant difference in muscle soreness 48 hours after exercise between all three groups (F (2.0, 21.0)=7.9, P = 0.003). A Bonferroni post hoc test was performed and similarly, there was no significant difference found between placebo and 4g of CM (P = 0.099); no significant difference between placebo and 8g of CM (P = 0.324); nevertheless, there was a significant difference between 4g of CM and 8g of CM (P = 0.002).

(Figure 4. The total level of muscle soreness recorded 24 and 48 hours following exercise for each week. Bars represent mean muscle soreness values. Error bars represent standard deviation.)Gastrointestinal Discomfort

Reporting’s of gastrointestinal discomfort were non-existent. In result, no direct measures were conducted.

Discussion

The purpose of the current study was to examine the effect of CM supplementation on athletic anaerobic performance during a bench press protocol, recently administered by Pérez-Guisado and Jakeman (2010). The primary finding of this randomised, double blind crossover design was that, in stark contrast to the hypothesis, there was no significant increase in total repetitions performed towards the end of each session when supplementing with CM. Similarly, the effect of CM on muscle soreness 24 and 48 hours post-exercise was found to be of insignificance in relation to a placebo, thus providing evidence to assume that CM has no effect on decreasing total muscle soreness experienced post-exercise. Moreover, there were no values reported for gastrointestinal discomfort experienced throughout the experiment.

Due to limited literature, the understandings for the proposed effects of CM on anaerobic performance remain inconclusive; supplying limited evidence towards the specific mechanisms for the understandings of CM’s effect as an ergogenic aid. However, recent findings postulate supporting evidence as towards its effect on aerobic performance that may help to rationalise its hypothetical effect on anaerobic performance. The proposed theoretical approach by Pérez-Guisado and Jakeman (2010), their understanding of CM, and its use as an ergogenic aid is considerably vague. Research provides significant findings in relation to increased levels of NO production through nitrate levels in the blood after a cycling stage (Sureda et al., 2010). It was evidenced that L-arginine levels and endothelial NO synthesis in plasma concentrations increased in the CM supplemented group (Sureda et al., 2010), thus supporting current literature (Béscos et al., 2012; Talbott, 2003) that recognises the effect of L-arginine on increasing levels of NO in the body. However, Hickner et al. (2006) proposed to increase L-arginine levels, resulting in increased NO production via oral L-citrulline ingestion. This theoretical standpoint is through the ability of L-citrulline to avoid uptake into the liver, offering a substance highly ecological for maintaining most of its ingested properties (Alvares et al., 2011; Osowska et al., 2004). Whereas its predecessor, L-arginine, lacks the ability to utilise high amounts, therefore providing little benefit as opposed to L-citrulline (Osowska et al., 2004).

Consequently, documentation suggests that L-arginine has no effect as an ergogenic aid, resulting for further research to understand its mechanisms entirely (Alvares et al., 2012; Alvares et al., 2011; Bloomer et al., 2010). In the study by Hickner et al. (2006), L-citrulline was thought to have impaired performance during aerobic exercise, suggesting that it may have no effect when supplemented alone. This can be explained through aforementioned research, advocating no increase in performance through L-arginine supplementation (Alvares et al., 2012; Alvares et al., 2011). Hence, suggesting there to be no increase in performance regardless of the increased uptake caused by L-citrulline. It is under the assumption that L-citrulline could increase NO production indirectly through its metabolic conversion pathway, however, current findings report little evidence to support its current effect upon anaerobic performance. Conversely, several studies reported a beneficial effect, when supplementing with the more recently explored CM (Giannesini et al., 2011; Pérez-Guisado and Jakeman, 2010; Sureda et al., 2010; Bendahan et al., 2002).

Bendahan et al. (2002) suggest that aerobic ATP production is probably related to a change in mitochondrial function and not because of increased levels of L-citrulline, nevertheless, findings support its proposed intervention at increasing the clearance of ammonium from the body and suggest that it can play a minor role in the facilitation of energy production and a potential aid for decreasing lactate production (Bendahan et al., 2001; Vanuxem et al., 1986). In the current study, total measures for muscle soreness were examined 24 and 48 hours following exercise. Findings shown in figure 4 demonstrate significantly lower reporting’s of muscle soreness after supplementation of 8g CM, however, highest recordings were reported under the influence of 4g CM. Due to the nature of the test and its focus on resistance training, there are many factors that could influence muscle soreness. However, the primary reason for muscle soreness could be due to the possibility of undergoing unaccustomed stress, which over time, decreases with the repeatability of an exercise (Brown, 2007). The explanation for why muscle soreness occurs is still relatively unknown; however the phenomenon known as repeated-bout effect (Brown, 2007) is potentially the main cause for the constant decrease of muscle soreness experienced over time, regardless of supplementation influence (McHugh et al., 1999). Therefore, In relation to the current study and the effect of CM on decreased muscle soreness, it could be contested that the reliability of the results could be regarded as of little significance.

In the current study, total repetitions performed during the last set, after ingestion of 8g CM increased statistically (11.88 ± 4.85); however no significant difference was found. Supplementing with 4g CM resulted in the lowest scores (9.75 ± 4.46) in comparison with placebo (10.25 ± 4.65). Few studies endeavour reasonable explanations based on scientific information found at a molecular level, as to why CM may have an effect upon human performance. Pérez-Guisado and Jakeman (2010) studied the impact of CM on anaerobic performance and explained potential pathways for the increased production of ATP, although they believe it is related to the impact CM has on buffering acidosis, lactate accumulation and hyperammonemia. Callis et al. (1991) suggest that CM stimulates hepatic ureogenesis and facilitates the renal reabsorption of bicarbonates, which may explain its anti-fatigue properties because of its protective effect against acidosis and ammonia poisoning, however, the present study indicates contrasting evidence (Figure 3). Pérez-Guisado and Jakeman (2010) believe this is the underlying reason for explaining CM’s effect on anaerobic performance, and suggest that during short aerobic sessions and lower intensity anaerobic exercise (longer rest periods), CM supplementation would be less effective.

Giannesini et al. (2011) and Bendahan et al. (2002) suggest that a potential reason for the increase in aerobic ATP production could be due to the role of malate and its affects through anaplerotic reactions in the TCA cycle. The mechanisms for this particular replenishment system are still not clear, however, it is known that malate plays an important role in the transfer from cystolic NADH into mitochondrial NADH (Eto et al., 1999). In the electron transport chain, NADH and FADH2 (flavin adenine dinucleotide) are oxidised to yield ATP in the mitochondria through glycolysis and other cystolic reactions (Wu et al., 2007). NADH and FADH2 are impermeable to the mitochondria; therefore two essential shuttle systems are required for the movement of cystolic NADH into the mitochondria, the glycerol phosphate shuttle and the malate-aspartate shuttle (most dominating shuttle in the liver and cardiac mitochondria) (Wu et al., 2007; Hedeskov, Capito and Thams, 1987; MacDonald, 1981). The current study viewed the impact of CM on anaerobic performance; yet, only a few studies have reported beneficial effects of CM supplementation under aerobic conditions (Giannesini et al., 2011; Sureda et al., 2010; Bendahan et al., 2002). Moreover, there are a very few studies (Pérez-Guisado and Jakeman, 2010) that provide sufficient evidence towards the effects of CM supplementation on anaerobic performance in humans.

The role that malate plays as an intermediate in the TCA cycle (under aerobic conditions), may provide sufficient rational towards the explanation for the results linked within the current study. While aerobic energy production is the leading pathway for ATP delivery throughout most exercise situations, anaerobic ATP delivery is also required at the onset of exercise and during intense exercise (Spriet, 2006). Malate has been shown to benefit ATP production under aerobic conditions as formerly mentioned in both human and animal studies (Wu et al., 2007; Bendahan et al., 2002), yet the performance of malate anaerobically remains elusive (Béscos et al., 2012). Additionally, the aforementioned mechanisms of malate and its essential role in the malate-aspartate shuttle could help to systematically recognise the fundamental weakness of its role anaerobically. For example, Wu et al. (2007) supplied significant documentation on the importance of malate in the oxidative pathway for the conversion of cystolic NADH into mitochondrial NADH. Bendahan et al. (2002) further supports this literature through its understanding of anaplerotic reactions which have been found by Wu et al. (2007) to accelerate the flux rate of the TCA cycle that would enhance glycolysis and resulting reactions. Thus, providing significant evidence to suggest that malate may not have any effect on anaerobic performance due to its primary role in the facilitation of oxidative ATP production.

The current study produced no side effects with CM supplementation; therefore further research is needed to provide adequate evidence to support findings by Pérez-Guisado and Jakeman (2010). However, current literature remains inconsistent in analysing reports of gastrointestinal discomfort (Sureda et al., 2010; Bendahan et al., 2002). Furthermore, studies suggest that high dosages of L-arginine may cause nausea, gastrointestinal discomfort and diarrhoea in humans (Collier, Casey and Kanaley, 2005) (Daly et al., 1988), while few studies have reported gastrointestinal discomfort in humans through L-citrulline supplementation (Grimble, 2007), leading into future considerations for examining the effects of supplementing alone (L-arginine and L-citrulline) or combined (CM) on gastrointestinal discomfort.

The present study indicates possible explanations for the ineffectiveness of CM supplementation on anaerobic performance, however due to the availability of resources, time constraints, and participant availability, limitations exist in regards to scientific measures that could further the validity of results. Additional considerations could include biological NO markers for the recognition of NO production (Sureda et al., 2010); duration of experiment with specific incremental increases of weight each week, based on findings supported by Brown (2007) and McHugh et al. (1999); as well as further indicators for the measurement of muscle soreness (Flann et al., 2011).

It can be concluded that, in healthy young men, contrary to the hypothesised increase in repetitions to muscular failure, there was a reduction in repetitions completed following the ingestion of 4g CM, one hour before exercise. Additionally, there was no significant difference reported across 4 and 8g of CM supplementation against a placebo; however an improvement in repetitions with 8g of CM during the last set is clearly shown. Muscle soreness experienced 24 and 48 hours after exercise provided conflicting evidence, resulting in no significant difference against placebo. All participants reported no feelings of gastrointestinal discomfort, suggesting that CM supplementation supports previous research into its safety/acceptability as a potential ergogenic aid. Further research is needed to assess the current use of CM as a means for improving anaerobic performance.

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Set 19.24178999999999998.05339000000000038.83882999999999971Week 1 (4)Week 2 (0)Week 3 (8)2425.37524.875Set 24.80327000000000045.20302000000000045.39179000000000031Week 1 (4)Week 2 (0)Week 3 (8)14.2513.7514.25Set 34.65218999999998944.46414000000000044.85320000000000021Week 1 (4)Week 2 (0)Week 3 (8)9.7510.2511.875

Supplement Group (g)

Total Number of Repetitions

24hr1.30465999999999992.03466000000000010.459619999999999971Week 1 (4)Week 2 (0)Week 3 (8)2.86249999999999981.6750.2374999999999999948hr0.661030000000000011.1963501Week 1 (4)Week 2 (0)Week 3 (8)1.56250.662499999999999980

Supplement (g)

Ratings of Muscle Soreness (VAS)