This study compared the effects of order of muscle groups’ exercised (larger to smaller muscles vs. smaller to larger muscles) on the acute levels of total testosterone, free testosterone and cortisol during resistance training (RT) sessions. Healthy male participants (n=8; age: 28.8 ± 6.4 years; body mass: 87.0 ± 10.6 kg; body height: 181.0 ± 0.7 cm; BMI: 26.5 ± 4.1) were randomly separated into two experimental groups. The first group (LG-SM) performed an RT session (3 sets of 10 repetitions and a 2 min rest period) of the exercises in following order: bench press (BP), lat pulldown (LP), barbell shoulder press (BSP), triceps pushdown (TP) and barbell cut (BC). The second group (SM-LG) performed an RT session in following order: BC, TP, BSP, LA, BP. Blood was collected at the end of the last repetition of each session. Control samples of blood were taken after 30 min of rest. Significant differences were observed in the concentrations of total testosterone (p < 0.05), free testosterone (p < 0.0001) and cortisol (p < 0.0001) after both RT sessions in comparison to rest. However, when comparing LG-SM and SM-LG, no significant differences were found. The results suggest that, while RT sessions induce an acute change in the levels of testosterone and cortisol, this response is independent of the order of exercising muscle groups.