Effects of moderate physical activity (90 min at 45-50% of maximal O2 uptake 2 times daily) and "high" (2.5 g protein. kg-1. day-1, n = 6) or "normal" protein intake (1.0 g protein. kg-1. day-1, n = 8) on the pattern and rate of 24-h macronutrient utilization in healthy adult men were compared after a diet-exercise-adjustment period of 6 days. Energy turnover (ET) was determined by indirect and direct (suit) calorimetry, and "protein oxidation" was determined by a 24-h continuous intravenous infusion of [1-13C]leucine. Subjects were in slight positive energy balance during both studies. Protein contributed to a higher (22 vs. 10%) and carbohydrate (CHO) a lower (33 vs. 58%) proportion of total 24-h ET on the high- vs. normal-protein intake. The highest contribution of fat to ET was seen postexercise during fasting (73 and 61% of ET for high and normal, respectively). With the high-protein diet the subjects were in a positive protein (P < 0.001) and CHO balance (P < 0.05) and a negative fat balance (P < 0.05). The increased ET postexercise was not explained by increased rates of urea production and/or protein synthesis.