Obesity is undoubtedly caused by a chronic positive energy balance. However, the early metabolic and hormonal responses to overeating are poorly described. This study determined glycaemic control and selected gut hormone responses to nutrient intake before and after 7 d of high-fat overfeeding. Nine healthy individuals (five males, four females) performed a mixed meal tolerance test (MTT) before and after consuming a high-fat (65 %), high-energy (+50 %) diet for 7 d. Measurements of plasma glucose, NEFA, acylated ghrelin, glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP) and serum insulin were taken before (fasting) and at 30-min intervals throughout the 180-min MTT (postprandial). Body mass increased by 0·79 (sem 0·14) kg after high-fat overfeeding (P<0·0001), and BMI increased by 0·27 (sem 0·05) kg/m2 (P=0·002). High-fat overfeeding also resulted in an 11·6 % increase in postprandial glucose AUC (P=0·007) and a 25·9 % increase in postprandial insulin AUC (P=0·005). Acylated ghrelin, GLP-1 and GIP responses to the MTT were all unaffected by the high-fat, high-energy diet. These findings demonstrate that even brief periods of overeating are sufficient to disrupt glycaemic control. However, as the postprandial orexigenic (ghrelin) and anorexigenic/insulintropic (GLP-1 and GIP) hormone responses were unaffected by the diet intervention, it appears that these hormones are resistant to short-term changes in energy balance, and that they do not play a role in the rapid reduction in glycaemic control.