The elevated cerebral serotonin (5-hydroxytryptamine) level is one of the mechanisms that contribute to the central nervous system fatigue during exercise (Newsholme and Blomstrand, 2006). Serotonin, a is associated with the feeling of lethargy and tiredness that may contribute to the loss of central drive and motivation (Davis and Bailey, 1997). This hypothesis is supported by several human and animal studies. The cerebral uptake of tryptophan, the precursor for serotonin synthesis, was significantly increased in humans during 3-hr cycling (Blomstrand et al., 2005). In addition, cerebral serotonin synthesis was elevated after treadmill running in rats (Chaouloff, 1997). The running time to exhaustion was significantly decreased after the administration of a serotonergic agonist, while it was significantly improved when given a serotonergic antagonist in rats (Bailey et al., 1993).
The rate of cerebral serotonin synthesis is regulated by the transport of plasma tryptophan across the bloodbrain barrier (Sharp et al., 1992). The ability of branchedchain amino acids (BCAA) to compete with tryptophan for crossing the blood brain barrier through the same transporter has provoked the hypothesis that the supplementation of these amino acids could reduce cerebral serotonin synthesis and prevent central fatigue during prolonged exercise (Blomstrand et al., 1997; Fernstrom, 2005). Indeed, the administration of BCAA prevented exercise-induced serotonin release in rat hippocampus (Gomez-Merino et al., 2001). Human studies have also shown that oral supplementation of BCAA could reduce ratings of perceived exertion and mental fatigue in maximal exercise (Blomstrand et al., 1997) and improve cognitive function after a 30-km cross-country race through reduced plasma tryptophan/BCAA ratio (Hassmen et al., 1994). However, except one study undertaken in warm conditions (Mittleman et al., 1998), most studies showed that BCAA supplementation had no effect on endurance performance (Blomstrand et al., 1995; 1997; Struder et al., 1998; van Hall et al., 1995).
One possible explanation for the lack of ergogenic effect of BCAA supplementation is the accompanied excess hyperammonemia resulted from the oxidation of these amino acids (MacLean and Graham, 1993; MacLean et al., 1994, 1996; Meeusen et al., 2006; Struder et al., 1998). It has been shown that cerebral uptake and accumulation of ammonia (NH3) was increased in humans during prolonged exercise (Nybo et al., 2005), which could induce central fatigue by alterations of cerebral energy metabolism and neurotransmission, and signaling pathways within the neuron (Wilkinson et al., 2010). Therefore, we hypothesized that incorporating arginine and citrulline with BCAA could improve endurance exercise N[H.sub.3] N[H.sub.3] production and reducing plasma tryptophan/BCAA ratio.
Both arginine and citrulline could reduce exercise-related accumulations of NH3 by increasing the urea cycle (Curis et al., 2005; Schaefer et al., 2002) and nitric oxide (NO) biosynthesis (Clarkson et al., 1996; Curis et al., 2005). Citrulline is more potent because of its high bio-availability (Rouge et al., 2007). It has been revealed that citrulline supplementation could increase plasma urea concentration and NO production (Sureda et al., 2010), while suppressing the exercise-induced hyperammonemia (Takeda et al., 2011) in prolonged exercise. Moreover, a combined supplementation of citrulline, arginine, and ornithine reduced plasma ammonia concentration after a single bout of exhaustive exercise in rats (Meneguello et al., 2003).
The purpose of this study was to investigate the combined supplementation of BCAA, arginine, and citrulline on endurance performance in two consecutive days in trained runners. The majority of previous studies investigating the alleviation of central fatigue in endurance exercise used a single bout of exercise. From our previous results, the effect of BCAA and arginine supplementation appears to be effective on the second consecutive day of intermittent high-intensity exercise, when the central fatigue is more apparent, in well-trained athletes (Chang et al., 2015). It is common for endurance athletes to participate in more than one event in a competition. The athletes would race in two or more consecutive days, which would lead to the accumulation of fatigue. However, to our best knowledge, the nutritional strategy to alleviate central fatigue and improve endurance performance in consecutive days has not been investigated. The use of race-like environment in performance measurement would make the results more applicable to real competitions. Each trial contained two consecutive days of exercise, with a 5000 meter (m) time trial on the first day, and a 10000 m time trial on the second day.
Thirteen endurance runners (10 male and 3 female) were originally recruited from the track and field team in National Taiwan University of Sport, Taichung, Taiwan. The subjects have trained and competed in events ranging from 1500 m to 10000 m. Three of them withdrew from the study because of sickness or injuries unrelated to the supplements and tests. The remaining 10 participants (7 male and 3 female) have been participating in endurance training for 7.3 [+ or -] 0.9 years and competed at the national level. The 7 male participants have the age of 20.6 [+ or -] 1.1 years, the height of 1.72 [+ or -] 0.08 m, the weight of 57.03 [+ or -] 4.92 kg, body mass index of 19.14 [+ or -] 1.35 kg x [m.sup.-2], and the body fat of 12.9 [+ or -] 2.1%. The 3 female participants have the age of 22.7 [+ or -] 2.3 years, the height of 1.58 [+ or -] 0.06 m, the weight of 46.80 [+ or -] 4.57 kg, the body mass index of 18.67 [+ or -] 0.58 kg x [m.sup.-2], and the body fat of 18.6 [+ or -] 0.8%. The body composition was measured by bioelectrical impedance analysis (IOI 353, Jawon Medical, Gyeongsan-si, Korea). The exclusion criteria included major cardiovascular disease risks, musculoskeletal injuries, upper respiratory infection, smoking, and consumption of any medicine or protein/amino acids supplement in the past 3 months. The participants were advised to maintain their regular training schedule and...