Purpose: The present study examined the effects of pre-exercise high and low GI meals on substrate metabolism and appetite in inactive middle-aged Japanese women. Methods: Fifteen inactive middle-aged women completed two trials in a random order. Participants consumed a high GI (HGI) or low GI (LGI) breakfast at 0900 and rested until 1100. Thereafter, participants performed a 60-minute walk at 50% of estimated maximum oxygen uptake (1100-1200) and rested until 1300. Expired gas samples were collected every 30 minutes for 15 minutes prior to walking, and further samples were collected continuously throughout the walking and post-walking periods. Blood samples and ratings of subjective appetite were collected every 30 minutes, except during walking, throughout the trial. Results: The cumulative fat oxidation was higher whereas the cumulative carbohydrate oxidation was lower during walking in the LGI trial than the HGI trial (Trial, P = 0.026, P = 0.012, respectively). There was a tendency toward a higher overall appetite score in the LGI trial than the HGI trial (Trial, P = 0.06). Conclusion: These findings indicate that consuming a LGI breakfast have favourable effects on fat oxidation during subsequent walking and appetite in inactive middle-aged Japanese women.