It is unclear how timing of exercise relative to meal ingestion influences substrate balance and metabolic responses. The present study aimed to compare the effects of exercise performed before or after breakfast on fat balance and postprandial metabolism. A total of ten sedentary overweight men (aged 28·1 (sem 10·7) years, BMI 29·0 (sem 2·8) kg/m2) underwent three trials in random order involving: (1) performing no exercise (CON), or walking for 60 min at 50 % maximal O2 uptake either (2) before (Ex-Meal) or (3) after (Meal-Ex) consuming a standardised breakfast meal. In each trial an ad libitum lunch was provided 3·5 h after breakfast. Substrate utilisation was assessed by indirect calorimetry and blood was taken at regular intervals over an 8·5 h observation period. At the end of the observation period, fat balances in the Ex-Meal ( − 1043 (sem 270) kJ) and Meal-Ex ( − 697 (sem 201) kJ) trials were both significantly lower than CON (204 (sem 165) kJ) and fat balance in the Ex-Meal trial was significantly lower than in the Meal-Ex trial (all P <0·0001). Compared with the CON trial, the 8·5 h postprandial TAG response was only significantly lowered in the Ex-Meal trial ( − 17 %, P =0·025) and not in the Meal-Ex trial ( − 11 %, P =0·20). Both the Ex-Meal and Meal-Ex trials showed significantly lowered insulin responses relative to the CON trial (by 19 and 24 %, respectively, P <0·01 for both). There were no differences in lunch energy intake between trials. The present findings suggest that there may be an advantage for body fat regulation and lipid metabolism in exercising before compared with after breakfast. However, further study is needed to determine whether the present findings extend over the long term under free-living conditions.