Technical Abstract: Background: The results of short-term studies in humans suggest that, compared to glucose, acute consumption of fructose leads to increased postprandial energy expenditure (PPEE) and carbohydrate oxidation and decreased postprandial fat oxidation. Objective: To determine the relative effects of these dietary sugars on energy expenditure and substrate utilization following sustained consumption. Design: As part of a double-blinded parallel arm study, older, overweight and obese male and female subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Energy expenditure and substrate utilization were assessed using indirect calorimetry at baseline and following 10 weeks of intervention. Results: Consumption of both sugars led to significant decreases in resting and postprandial metabolic rate despite comparable increases in weight. Consumption of fructose, but not glucose, led to significant decreases in postprandial fat oxidation and significant increases in postprandial carbohydrate oxidation and RQ. In subjects consuming fructose, the observed changes in substrate utilization were correlated with increases in de novo lipogenesis (DNL) that were reported previously. In addition, the observed decreases in postprandial fat oxidation were correlated with previously reported increases in intra-abdominal fat area. Conclusions: These findings suggest that sustained consumption of fructose- and glucose-sweetened beverages leads to a decrease in PPEE that is independent of changes in total body mass, and that consumption of fructose specifically leads to significant changes in postprandial substrate utilization that are associated with increases in hepatic DNL.