When previously sedentary men and women follow exercise training programs with ad libitum feeding, men lose body fat but women do not. The purpose of this study was to evaluate whether this observation could be related to sex differences in the way energy regulating hormones and appetite perception respond to exercise. Eighteen (9M,9W) overweight/obese individuals completed 4 bouts of exercise with energy added to the baseline diet to maintain energy balance (BAL), and 4 bouts without energy added to induce energy deficit (DEF). Concentrations of acylated ghrelin, insulin and leptin, and appetite ratings were measured in response to a meal after a no-exercise baseline and both exercise conditions. In men, acylated ghrelin area under the curve (AUC) was not different between conditions. In women, acylated ghrelin AUC was higher after DEF (+32%) and BAL (+25%), and change from baseline was higher than men (p<0.05). In men, insulin AUC was reduced (-17%) after DEF (p<0.05), but not BAL. In women, insulin AUC was lower (p<0.05) after DEF (-28%) and BAL (-15%). Leptin concentrations were not different across conditions in either sex. In men, but not in women, appetite was inhibited after BAL relative to DEF. The results indicate that, in women, exercise altered energy regulating hormones in a direction expected to stimulate energy intake, regardless of energy status. In men, the response to exercise was abolished when energy balance was maintained. The data are consistent with the paradigm that mechanisms to maintain body fat are more effective in women.