Few studies have examined the influence of pre-exercise meals with different glycaemic indices (GIs) on substrate oxidation and non-homeostatic appetite (i.e. food reward) in adults of various ages and ethnicities. We aimed to examine the effects of pre-exercise high and low GI meals on substrate oxidation and food reward in middle-aged Japanese women. This randomised crossover trial included fifteen middle-aged women (aged 40⋅9 ± 6⋅5 years, mean ± sd). The participants consumed a high or low GI breakfast at 09.00 and rested until 11.00. Thereafter, participants performed a 60-min walk at 50 % of their estimated maximum oxygen uptake (11.00–12.00) and rested until 13.00. Expired gas samples were collected every 30 min prior to walking, and samples were collected continuously throughout the walking and post-walking periods. Blood samples and subjective appetite ratings were collected every 30 min, except during walking. The Leeds Food Preference Questionnaire in Japanese (LFPQ-J) was used to assess food reward at 09.00, 10.00, and 13.00 h. The cumulative fat oxidation during exercise was higher in the low GI trial than in the high GI trial (P = 0⋅03). The cumulative carbohydrate oxidation during walking was lower in the low GI trial than in the high GI trial (P = 0⋅01). Trial-by-time interactions were not found for any food-reward parameters between trials. Low GI meals elicited enhanced fat oxidation during a subsequent 60-min walk in middle-aged women. However, meals with different GIs did not affect food reward evaluated over time in the present study.