It is well known that substrate oxidation rates are increased by exercise. The present study had two main objectives: firstly, to examine the effect of a single exhaustive exercise session on post-exercise substrate oxidation and energy expenditure; and secondly, to determine the differences between athletes and non-Athletes. Material and methods: Eighteen healthy male athletes (mean ± SD age; 19.38 ± 2.26 years, VO2max; 60.57 ± 3.90 ml · kg-1 · min-1, n = 8) and non-Athletes (age; 20.30 ± 1.26 years, VO2max; 44.97 ± 5.43 ml · kg-1 ·min-1, n = 10) volunteered to participate in the study. After an overnight fast, subjects performed a single sprint exercise session on a cycle ergometer with individual loads (0.075 kg per body weight) until volitional exhaustion. Energy expenditure (EE) and the substrate oxidation rate were measured at rest and during the post-exercise recovery period using indirect calorimetry. Results: Exhaustive exercise significantly increased post-exercise fat oxidation, energy expenditure and contribution of fat to EE (p < 0.05). Also, it significantly decreased post-exercise carbohydrate (CHO) oxidation and the contribution of CHO to EE (p < 0.05). However, the changes in the substrate oxidation rate and EE after the exercise test were not different between the groups (p > 0.05). Conclusions: The study results suggest that a single short-duration exhaustive exercise session causes a higher fat oxidation rate during recovery than at rest, whereas training status did not affect this situation