The ability to switch fuels for oxidation in response to changes in macronutrient composition of diet (metabolic flexibility) may be informative of the individual susceptibility to weight gain. Seventy-nine healthy, weight-stable subjects underwent 24-h assessments of energy expenditure and respiratory quotient (RQ) in a whole-room calorimeter during energy balance (EBL; 50% carbohydrate, 30% fat) and then during 24-h fasting and three 200% overfeeding diets in a crossover design. Metabolic flexibility was defined as the change in 24-h RQ from EBL during fasting and standard (SOF: 50% carbohydrate, 30% fat), high-fat (FNP: 60% fat, 20% carbohydrate), and high-carbohydrate (CNP: 75% carbohydrate, 5% fat) overfeeding diets. Free-living weight change was assessed after 6 and 12 months. Compared to EBL, RQ decreased on average by 9% during fasting and by 4% during FNP, while increasing by 4% during SOF and by 8% during CNP. Smaller decrease in RQ, reflecting smaller increase in lipid oxidation rate, during FNP but not during other diets, predicted greater weight gain at both 6 and 12 months. An impaired metabolic flexibility to acute, high-fat overfeeding identify individuals prone to gain weight, indicating that the individual capacity to oxidize dietary fat is a metabolic predictor of weight change.