BACKGROUND:The purpose of the present study was to examine the effects of time-ofday-strength training on plasma testosterone (T) and cortisol (C) concentrations in male amateur athletes. METHODS: Forty five male athletes (age, 21.2±0.3 years; height, 1.75±0.14 m; weight, 72±1.52 kg) participated in the present study. They were subjected to strength training of the knee extensor and flexor muscles. Following this, they were randomly assigned into three groups: morning strength training group (MSTG, 07:00-08:00 hours, n=15), afternoon strength training group (ASTG, 16:00-17:00 hours, n=15) and morning and afternoon strength training group (MASTG, 07:00-08:00 and 16:00-17:00 hours, n=15). The concentration of T and C for each athlete was collected in all three conditions in the morning, noon and afternoon, before and after 8 weeks of strength training. The effects of group, time of the day and pre-to post-training were verified by a 3-way analysis of variance with repeated measures. RESULTS: Our results indicate that strength training induces an increase of plasma T/C ratio in both groups of athletes who have always trained for only one session per day mainly in the afternoon compared with the MASTG (P < 0.05). However, a reduction of the plasma T/C ratio was observed in the athletes who have always trained successively in the morning and in the afternoon (P < 0.05). Plasma T/C ratio exhibit circadian rhythmicity in all groups showing a low level in the morning, whereas in the afternoon an increased value was more noted (P < 0.05). CONCLUSIONS: From an applied perspective, this study suggests that amateur athletes should train at the afternoon to maximize their performance gain.