Background: We previously demonstrated that short-term (24h) changes in carbohydrate (CARBOX) or fat oxidation rates in response to overfeeding diets with different macronutrient content are highly correlated within an individual, suggesting the existence of human metabolic phenotypes (carbohydrate vs. fat oxidizers). Gut hormones have a role in feeding and substrate oxidation, thus we investigated if the changes in gut hormones concentration during overfeeding diets or fasting explain the degree of metabolic flexibility in healthy humans.Methods: While residing in our clinical research unit, 67 healthy, weight-stable volunteers (37±10y, BMI: 26±4 kg/m2, body fat: 28±10%; mean±SD, 54 men) with normal glucose regulation had 24-h EE measurements in a whole-room indirect calorimeter during energy balance (EB, diet: 50% carbohydrate, 20% protein), three overfeeding diets, and fasting in a crossover design. The overfeeding diets (200% of weight-maintaining energy requirements) included diets with 20%-protein [50%-carbohydrate (SOF), 75%-carbohydrate (CNP), and 60%-fat (FNP)]. Metabolic flexibility was determined by the difference between respiratory quotient (RQ) during overfeeding/fasting from EB conditions. Plasma GLP-1, PYY, PP, and total ghrelin concentrations were measured by ELISA after an overnight fast the morning of and after each diet. Results: PYY increased after all overfeeding diets (3.4±13.1, 7.0±16.2, and 7.6±14.4 pg/mL; all p<0.05, SOF, CNP and FNP respectively) and decreased with fasting (-11.6±14.2 pg/mL; p<0.001). GLP-1 increased after fasting (2.7±5.8 pg/mL; p<0.001) and FNP (1.3±4.5 pg/mL; p=0.02) and decreased with CNP (-0.9±6.3 pg/mL; p=0.04). PP only increased after fasting (92.8±133.4 pg/mL; p<0.001), while ghrelin decreased with SOF (-31.3±108 pg/mL; p=0.02) and CNP (-43.1±120 pg/dL; p=0.005) but not with FNP (p=0.51). After adjustment for body composition and other known EE determinants, no hormonal changes were associated with the change in 24-h EE in any diet (all p>0.05); however, during CNP greater decreases in GLP1, PYY, and Ghrelin were associated with less increase in 24-h RQ (all r>0.25; all p<0.05) while greater decrease in PP was associated with larger increase in 24-h RQ (r=-0.31, p=0.01). Specifically, greater increase in CARBOX during CNP was positively associated with the changes in GLP-1 (r=0.30; p=0.02) and Ghrelin (r=0.32, p=0.01). In a linear model, only the change in GLP-1, PP, and Ghrelin concentrations during CNP were independent predictors of the change in RQ [β=0.003; -0.0001; and 0.000006 per 1 pg/mL increase each, p=0.008; 0.02 and 0.02 respectively], after accounting for age and sex. Conclusion: In a carbohydrate rich environment, individuals with normal glucose regulation who maintain GLP-1 and ghrelin levels are better adapted to metabolize this type of diet.