The objective of this study was to determine the effect of increased physical activity on subsequent sleeping energy expenditure (SEE) measured in a whole room calorimeter under differing levels of dietary fat. We hypothesized that increased physical activity would increase SEE. Six healthy, young men participated in a randomized, single blind, crossover study. Subjects repeated an eight-day protocol under four conditions separated by at least 7 days. During each condition, subjects consumed an isoenergetic diet consisting of 37% fat, 15% protein and 48% carbohydrate for the first four days and for the following four days, SEE and energy balance were measured in a respiration chamber. The first chamber day served as a baseline measurement and for the remaining three days, diet and activity were randomly assigned as high fat/exercise, high fat/sedentary, low fat/exercise, or low fat/sedentary. Energy balance was not different between conditions. When the dietary fat was increased to 50%, SEE increased by 7.4% during exercise (P<0.05) relative to being sedentary (baseline day) but SEE did not increase with exercise when fat was lowered to 20%. SEE did not change when dietary fat was manipulated under sedentary conditions. Physical activity causes an increase in SEE when dietary fat is high (50%), but not when dietary fat is low (20%). Dietary fat content influences the impact of post-exercise induced increases in sleeping energy expenditure. This finding may help explain the conflicting data regarding the effect of exercise on energy expenditure.