Acute phosphatidylserine (PS) or branched-chain amino acids (BCAA) supplements alone may have an adrenocorticotropic hormone, cortisol suppressive effect and increase the testosterone/cortisol ratio, but the associated effect of these supplements during a period of high-intensity physical stress is not yet known. The study investigated the effects of chronic PS supplementation alone and combined with BCAA during high-intensity interval training (HIIT) on training volume tolerance, anabolic-catabolic balance and stress biomarkers in rats. Thirty-three rats were separated into: placebo (PLA, n=11), PS alone (n=11) and combined with BCAA (PSBCAA, n=11). Groups performed swimming sessions of HIIT (5 series × 1 min × 1 min recovery; external load equivalent to 13% of body mass) and nine recovery sessions of moderate-intensity training (30 min at 5% of body mass) alternately. One-way ANOVA was used to compare biochemical variables and two-way ANOVA was calculated to compare training volume. Training volume performed (TVP) was higher in first, fourth, fifth, sixth, and eighth HIIT sessions in the PS group in comparison to PLA (P<0.05). TVP was higher in the fourth session in PSBCAA compared to PLA. There were no differences in TVP during the sessions between PS and BCAA groups. Creatine kinase (CK) was lower in PSBCAA in comparison to PS alone (P=0.03) and PLA (P=0.04) after the experimental period. Testosterone concentration was enhanced in PSBCAA group compared to PLA (P=0.01); testosterone/corticosterone ratio was higher in PSBCAA compared to PS (P=0.05) and PLA (P=0.004) after protocol. PS combined with BCAA increases testosterone concentration and testosterone/corticosterone ratio, demonstrating an enhancement of anabolic state in trained rats.