Objective: To investigate whether the ‘overnight second-meal effect’ results in altered substrate oxidation during the postprandial period following breakfast and subsequent sub-maximal exercise in women. Subjects/Methods: Seven recreationally active women were recruited for the study. In each trial, participants were provided with their evening meal on day 1, which was composed of either high glycaemic index (HGI) or low glycaemic index (LGI) carbohydrates (CHO). On day 2, participants were provided with a standard HGI breakfast and then performed a 60-min run at 65% O2 max 3?h later. Results: The incremental area under the curve (IAUC) for plasma glucose concentrations during the postprandial period following breakfast was greater in the HGI trial compared to the LGI trial (P<0.01). Similarly, the IAUC for serum insulin concentrations was greater in the HGI trial than the LGI trial (P<0.05). No differences in plasma free-fatty acids (FFA) or plasma glycerol concentrations were found between trials during the postprandial period. During subsequent exercise, there were no significant differences in substrate metabolism. Conclusion: The glycaemic index of an evening meal does not alter substrate oxidation at rest following breakfast or during subsequent submaximal exercise in women. This study provides further evidence for the overnight second-meal effect on glycaemic responses following a LGI mixed evening meal.