Fluctuations in substrate preference and utilization across the circadian cycle may be influenced by the degree of physical activity and nutritional status. In the present study, we assessed these relationships in control mice and in mice from a line selectively bred for high voluntary wheel-running behavior, either when feeding a carbohydrate-rich/low-fat (LF) or a high-fat (HF) diet. Housed without wheels, selected mice, and in particular the females, exhibited higher cage activity than their non-selected controls during the dark phase and at the onset of the light phase, irrespective of diet. This was associated with increases in energy expenditure in both sexes of the selection line. In selected males, carbohydrate oxidation appeared to be increased compared to controls. In contrast, selected females had profound increases in fat oxidation above the levels in control females to cover the increased energy expenditure during the dark phase. This is remarkable in light of the finding that the selected mice, and in particular the females showed higher preference for the LF diet relative to controls. It is likely that hormonal and/or metabolic signals increase carbohydrate preference in the selected females, which may serve optimal maintenance of cellular metabolism in the presence of augmented fat oxidation. (C) 2010 Elsevier Inc. All rights reserved.