Serotonin, a neurotransmitter synthesized from tryptophan(TRP), has been proposed to play a key role in central fatigue. Here, we examined whether TRP itself and/or its two metabolites, kynurenic acid (KYNA) and quinolinic acid (QUIN), are involved in central fatigue. Experiments were conducted using Sprague-Dawley rats (SDR, 200-250 g, n = 21) and Nagase analbuminemic rats (NAR, 210-255 g, n = 3). Central fatigue was assessed by treadmill running and a Morris water-maze test. First, a microdialysis guide cannula (CMA/12) was inserted into the left striatum under anesthesia with sodium pentobarbital (25 mg/kg i.p.; Abbott Laboratories, USA), and microdialysis (Yamamoto et al, 2012) was used to collect samples for measurement of extracellular concentrations of TRP, serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) in real time as rats (SDR and NAR) ran to exhaustion on the treadmill. Rats (SDR) were first treated with either saline or Branched-chain amino acids (BCAA, valine:leucine:isoleucine, 5:3:2, 250 mg/kg i.p.) 30 min before the exhaustion test. Second, the rats (another group of SDR) were trained in four daily sessions for 10 consecutive days on a Morris water-maze (a circular water tank 147 cm in diameter fitted with a refuge platform 250 mm in height). Rats were tested 6-7 days after injection with 180 nmol QUIN or QUIN plus 20 nmol KYNA into the hippocampus CA3-region through a guide cannula (CMA/12). All animal procedures were conducted in accordance with guidelines of the Japanese Neuroscience Society for animal experiments, and sanctioned by the animal-research ethics committee of Tezukayama University. The concentration of TRP secreted into the extracellular space of the striatum was higher during fatigue, and quickly returned to basal (free moving) levels with recovery from fatigue. Intracerebral TRP levels were directly associated with the time-to-exhaustion and were shown to be easily reduced with BCAA treatment. Compared with the saline group (133 ± 6.3 min), time-to-exhaustion for the BCAA group was clearly prolonged via the dramatic reduction in extracellular TRP caused by BCAA treatment (187 ± 21.5 min) (t = 2.404, d.f. = 7, p < 0.05). Moreover, return to basal TRP levels was associated with recovery from exhaustion, with real-time release of TRP in extracellular fluid showing that this response was sensitive and specific. Time-to-exhaustion was shorter in NAR, which maintain a higher extracellular level of striatal TRP than do SDR. Impaired memory performance in a water maze task after TRP treatment was attributable to high levels of KYNA and QUIN in the hippocampus acting synergistically on N-methyl-D-aspartic acid receptors. Our findings demonstrate that the increase in fatigue that occurs because of elevated brain TRP can be further amplified with synthetic KYNA and QUIN.