Objective:The roles that energy expenditure (EE) and nutrient oxidation play in a predisposition for weight gain in humans remains unclear.Subjects:We measured EE and respiratory exchange ratio (RER) in non-obese obesity-prone (OP; n=22) and obesity-resistant (OR; n=30) men and women following a eucaloric (EU) diet and after 3 days of overfeeding (1.4 × basal energy).Results:Twenty-four hour EE, adjusted for fat-free mass and sex, measured while consuming a EU diet was not different between OP and OR subjects (2367±80 vs 2285±98 kcals; P=0.53). Following overfeeding, EE increased in both OP and OR (OP: 2506±63.7, P<0.01; OR: 2386±99 kcals, P<0.05). Overfeeding resulted in an increase in 24-hour RER (OP: 0.857±0.01 to 0.893±0.01, P=0.01; OR: 0.852±0.01 to 0.886±0.01, P=0.005), with no difference between groups in either the EU or overfeeding conditions (P>0.05). Nighttime RER (∼10pm–6:30am) did not change with overfeeding in OR (0.823±0.02 vs 0.837±0.01, P=0.29), but increased significantly in OP subjects (0.798±0.15 to 0.839±0.15, P<0.05), suggesting that fat oxidation during the night was downregulated to a greater extent in OP subjects following a brief period of overfeeding, as compared with OR subjects who appeared to maintain their usual rate of fat oxidation. Protein oxidation increased significantly in both OP (P<0.001) and OR (P<0.01) with overfeeding, with no differences between OP and OR.Conclusion:These results support the idea that overfeeding a mixed diet results in increases in EE and RER, but these increases in EE and RER are likely not responsible for obesity resistance. Adaptive responses to overfeeding that occur during the night may have a role in opposing weight gain.