The central nervous system plays a crucial role in the development of physical fatigue. The purpose of this study is to investigate the effect of combined supplementation of branched-chain amino acids (BCAA) and arginine on intermittent sprint performance in simulated handball games on 2 consecutive days. Methods: Fifteen male and seven female handball players consumed 0.17 g/kg BCAA and 0.04 g/kg arginine together (AA trial), or placebo (PB trial) before exercise. Each trial contained two 60-min simulated handball games on consecutive days. The game was consisted of 30 identical 2-min blocks and a 20 m all-out sprint was performed at the end of each block. The performance, measured by percentage changes of sprint time between day 1 and 2, was significantly better in the AA trial (first half: AA trial: -1.34±0.60%, PB trial: -0.21±0.69%; second half: AA trial: -1.68±0.58%, PB trial: 0.49±0.42%). The average ratings of perceive exertion throughout the 2-day trial was significantly lower in the AA trial (14.2±0.3) than the PB trial (15.1±0.4). Concurrently, post-exercise tryptophan/BCAA ratio on both days in the AA trial was significantly lower than the baseline. This study showed that BCAA and arginine supplementation could improve performance in intermittent sprints on the second consecutive day of simulated handball games in well-trained athletes by potentially alleviating central fatigue.