This study was designed to determine endocrine responses during 2 days of strenuous resistance training. Ten healthy men performed resistance training twice a day for two successive days to induce acute fatigue (excessive physical stress). The resistance training consisted of four exercises for the lower body in the morning and seven exercises for the upper body in the afternoon. Maximal isometric and isokinetic strengths were measured from day 1 (before the training period) to day 3 (after the training period). Fasting blood samples were taken on days 1–3. Maximal isometric and isokinetic strengths significantly decreased with two successive days of training (P<0·05), with significant increases in serum creatine phosphokinase and myoglobin concentrations (P<0·05). Significant reductions in the fasting concentrations of serum insulin-like growth factor-1, free testosterone, insulin and high-molecular-weight adiponectin were observed on day 3 (P<0·05), whereas there were no changes in the serum cortisol concentration or the free testosterone/cortisol ratio. Plasma active ghrelin and serum leptin concentrations decreased by −20·7 ± 2·8% and −29·6 ± 4·1%, respectively (P<0·05). Two days strenuous resistance training significantly affects the profiles of anabolic hormone and endocrine regulators of appetite and energy balance, such as ghrelin and leptin. The present findings suggest that decreased ghrelin and leptin concentrations might reflect excessive physical stress and may be early signs of accumulated fatigue.