OBJECTIVE To investigate if the improved glucose tolerance previously observed at breakfast following an evening meal with boiled barley kernels derives from colonic events related to the fermentation of the elevated amounts of indigestible carbohydrates present and/or from the low-GI features. METHODS Twenty healthy volunteers aged 19-30 years. METHODS High-GI white wheat bread (WWB), WWB+barley dietary fibre (DF) corresponding to the DF content of barley kernels, low-GI spaghetti+ barley DF, spaghetti+double amounts of barley DF (2(*)DF), spaghetti+oat DF, or whole grain barley flour porridge, were provided as late evening meals. At a subsequent standardised WWB breakfast, B-glucose, s-insulin, p-SCFA, p-FFA, and breath hydrogen (H(2)) were measured. RESULTS The B-glucose response (incremental areas under the curves (IAUC) 0-120 min and total areas under the curves 0-180 min) to the standardized breakfast was significantly lower after consuming spaghetti+2*DF in the evening compared with barley porridge (P=0.012). The spaghetti+2*DF meal also resulted in the highest breath H(2) excretion (P<0.02). The glucose IAUC (0-120 min) after the standardized breakfast was positively correlated to fasting p-FFA (r=0.29, P<0.02), and the total glucose area (0-180 min) was negatively correlated to the p-propionate level (0-30 min) (r=-0.24, P<0.02). CONCLUSIONS The prolonged digestive and absorptive phase per se, like with a low-glycaemic index (GI) spaghetti evening meal, did not induce overnight benefits on glucose tolerance. Addition of barley DF in high amounts (2*DF) was required to improve overnight glucose tolerance. The correlations observed between glycaemia and p-propionate implicate colonic fermentation as a modulator of glucose tolerance through a mechanism leading to suppressed free fatty acids levels. It is proposed that the overnight benefits on glucose tolerance previously reported for boiled barley kernels is mediated through colonic fermentation of the prebiotic carbohydrates present in this product.