Changes in substrate oxidation with isoenergetic high-carbohydrate (HC) and high-fat (HF) diets in male nonathletic subjects, aerobically trained athletes, and weight-trained athletes were examined in a crossover study. A whole-room respiration chamber was used to measure 24-h energy expenditure (EE) and substrate oxidation with control, HC, or HF diets for 7 d. The nonathletic group had higher 24-h EE (P < 0.05), exercise EE (P < 0.03), and resting metabolic rate (P < 0.04) than did the aerobically trained athletes when these measurements were corrected for lean body mass. Fat oxidation was significantly correlated with lean body mass and diet. However, athletic status had no effect on substrate oxidation. Carbohydrate oxidation across groups increased acutely by 23% after 24 h of the HC diet (P < 0.0001). Carbohydrate balance increased significantly over time with the HC diet (P < 0.002) and decreased acutely after return to the control diet (P < 0.0001). With the HF diet, carbohydrate balance increased and was significantly different from balance with the control diet by day 7 (P < 0.03). Fat balance decreased significantly with both the HF (P < 0.04) and HC (P = 0.0075) diets by day 7. Carbohydrate oxidation correlated with carbohydrate intake with both the control (r = 0.61, P < 0.01) and HC diets (r = 0.59, P < 0.02), but not the HF diet. Fat oxidation was not correlated with fat intake. In conclusion, substrate oxidation in a respiration chamber is significantly affected by diet, but not by prior athletic training.