OBJECTIVES: To investigate the influences of inactivity and dietary macronutrient composition on energy and fat balance and to look for interactions between them. DESIGN: Two-day measurements of energy expenditure and substrate oxidation on five occasions; ad libitum food intake from diets of 35% and 60% energy as fat, with and without imposed activity, and a fixed overfeeding at 35% fat with free activity. SUBJECTS: Eight normal-weight male volunteers. MEASUREMENTS: Energy expenditure and substrate oxidation by indirect whole-body calorimetry, and macronutrient intakes from food consumption on ad libitum regimens. RESULTS: Subjects consumed the same energy, mean 11.6 MJ/d, regardless of activity level, on the 35% diet. Subjects consumed more energy on the 60% than the 35% diet, mean 14 vs 11.6 MJ/d. Inactivity induced a strong positive energy balance: 5.1 (60% diet), and 2.6 MJ/d (35% diet). Energy balance with activity was not significantly different between diets, nor significantly different from zero: 1.1 MJ/d (60% diet), and - 0.2 MJ/d (35% diet). When intentionally overfed, subjects failed to compensate by raising voluntary activity. CONCLUSION: Energy intake was not regulated over a 2-day period in response to either imposition of inactivity or a high-fat diet. Activity proved essential to the avoidance of significant positive energy balance.