Introduction. The time of exercise (morning and afternoon) can lead to changes in hormonal responses and exercise performance. Aim of Study. The current study aimed to investigate the effects of morning vs afternoon high-intensity interval training (HIIT) on testosterone (T), cortisol (C) and the testosterone/cortisol (T/C) ratio in active men. Material and Methods. Eleven active male students (aged: 19.0 ± 1.0 yrs., height: 177.5 ± 9.0 cm, weight: 70.6 ± 8.3 kg and BMI: 22.19 ± 1.88 kg/m2) completed two trials of the 40-m maximal shuttle run test (which incorporates 5 × 40 m shuttle sprints with 30 s between the start of each sprint), with seven days between the trials. All the trials were conducted indoors. Blood samples were taken before and immediately after each exercise session from the antecubital vein by repetitive venous puncture in a sitting position. Results. Data evidenced that the T concentration increased after HIIT in the afternoon (pre: 9.86 ± 0.42 vs post: 10.3. ± 0.61, P = 0.02). The significant difference was observed between pre-tests (T: 10.4 ± ± 0.67 vs 9.86 ± 0.42, P = 0.009; C: 898.38 ± 199.51 vs 355.53 ± 92.95, P = 0.001; T/C ratio: 0.012 ± 0.002 vs 0.03 ± 0.012, P = 0.001) and also post-test (T: 10.68 ± 0.53 vs 10.3. ± 0.61, P = 0.01; C: 990.64 ± ± 293.07 vs 452.73 ± 307.34, P = 0.001; T/C ratio: 0.011 ± 0.003 vs 0.056 ± 0.065, P = 0.04). Conclusions. It seems that performing high-intensity interval training (HIIT) in the afternoon may be more suitable in terms of the level of anabolic processes.