The central nervous system plays a crucial role in fatigue during endurance exercise. Branched-chain amino acids (BCAA) could reduce cerebral serotonin synthesis by competing with its precursor tryptophan for crossing the blood brain barrier. Arginine and citrulline could prevent excess hyperammonemia accompanied by BCAA supplementation. This study investigated the combination of BCAA, arginine, and citrulline on endurance performance in two consecutive days. Seven male and three female endurance runners ingested 0.17 g·kg^-1 BCAA, 0.05 g·kg^-1 arginine and 0.05 g·kg^-1 citrulline (AA trial) or placebo (PL trial) in a randomized cross-over design. Each trial contained a 5000 m time trial on the first day, and a 10000 m time trial on the second day. The AA trial had significantly better performance in 5000 m (AA: 1065.7 ± 33.9 s; PL: 1100.5 ± 40.4 s) and 10000 m (AA: 2292.0 ± 211.3 s; PL: 2375.6 ± 244.2 s). The two trials reported similar ratings of perceived exertion. After exercise, the AA trial had significantly lower tryptophan/BCAA ratio, similar NH₃, and significantly higher urea concentrations. In conclusion, the supplementation could enhance time-trial performance in two consecutive days in endurance runners, possibly through the inhibition of cerebral serotonin synthesis by BCAA and the prevention of excess hyperammonemia by increased urea genesis.

• The combined supplementation of BCAA, arginine, and citrulline could enhance performance in 5000 m and 10000 m in 2 consecutive days in competitive runners. The supplementation may be helpful in multi-day competitions.

• The supplemented BCAA may alleviate central fatigue, allowing the subjects to run faster at the same degree of perceived exertion.

• The hyperammonemia that is usually accompanied with BCAA supplementation may be prevented by arginine and citrulline through increased urea genesis.