The purpose of the present study was to investigate whether both glycemic index (GI) and fructose content of a pre-exercise meal would affect substrate utilization during subsequent brisk walking. Ten healthy young males completed 60 min of 46% [Formula: see text] brisk walking 2 h after they consumed one of three breakfasts: a low-GI meal without fructose (LGI), a low-GI meal including fructose (LGIF), and a high-GI meal without fructose (HGI). The calculated GI values for the three meals were 41, 39, and 72, respectively. Substrate utilization was measured using indirect respiratory calorimetry method. During the postprandial period, the incremental area under the blood response curve values of glucose and insulin were higher in the HGI trial, compared with those in the LGI and LGIF trials (HGI vs. LGI and LGIF: Glucose 223.6 ± 19.1 vs. 70.2 ± 7.4 and 114.1 ± 16.4 mmol min L(-1); Insulin 4257 ± 932 vs. 920 ± 319 and 1487 ± 348 mU min L(-1)). During exercise, substrate preference was distinct based on different pre-exercise carbohydrate meals. Higher fat and lower carbohydrate oxidation was observed in the LGI trial, whereas both the HGI and LGIF trials were characterized by higher carbohydrate and lower fat oxidation (LGI vs. LGIF and HGI: Carbohydrate 59.3 ± 2.4 vs. 69.8 ± 3.9 and 72.7 ± 3.9 g; Fat 22.7 ± 2.0 vs. 18.5 ± 1.7 and 17.6 ± 1.3 g; P < 0.05). In conclusion, the presence of fructose in a LGI breakfast resulted in similar substrate utilization during subsequent brisk walking with that induced by a HGI breakfast. It appears that both the GI and fructose content in a breakfast individually affect substrate utilization during subsequent moderate intensity exercise.