The purpose of the present study was to determine whether probiotic supplementation (Lactobacillus casei Shirota (LcS)) prevents diet-induced insulin resistance in human subjects. A total of seventeen healthy subjects were randomised to either a probiotic (n 8) or a control (n 9) group. The probiotic group consumed a LcS-fermented milk drink twice daily for 4 weeks, whereas the control group received no supplementation. Subjects maintained their normal diet for the first 3 weeks of the study, after which they consumed a high-fat (65 % of energy), high-energy (50 % increase in energy intake) diet for 7 d. Whole-body insulin sensitivity was assessed by an oral glucose tolerance test conducted before and after overfeeding. Body mass increased by 0·6 (se 0·2) kg in the control group (P< 0·05) and by 0·3 (se 0·2) kg in the probiotic group (P>0·05). Fasting plasma glucose concentrations increased following 7 d of overeating (control group: 5·3 (se 0·1) v. 5·6 (se 0·2) mmol/l before and after overfeeding, respectively, P< 0·05), whereas fasting serum insulin concentrations were maintained in both groups. Glucose AUC values increased by 10 % (from 817 (se 45) to 899 (se 39) mmol/l per 120 min, P< 0·05) and whole-body insulin sensitivity decreased by 27 % (from 5·3 (se 1·4) to 3·9 (se 0·9), P< 0·05) in the control group, whereas normal insulin sensitivity was maintained in the probiotic group (4·4 (se 0·8) and 4·5 (se 0·9) before and after overeating, respectively (P>0·05). These results suggest that probiotic supplementation may be useful in the prevention of diet-induced metabolic diseases such as type 2 diabetes.