Data obtained from studies conducted in animal models and humans suggest that gender affects the metabolic response to fasting. However, differences in body composition between males and females confound the interpretation of these studies, because increased adiposity itself alters the metabolic response to short-term fasting. We evaluated whole body lipid and glucose kinetics during basal (14-h fast) and short-term fasting (22-h fast) conditions in six women and six men who were matched for adiposity (24 ± 2 and 23 ± 2% body wt as fat, respectively). Substrate kinetics were measured by infusing stable isotope labeled tracers of glucose ([²H₂]glucose) and glycerol ([²H₅]glycerol). Basal glycerol rate of appearance (R_a) in plasma, an indicator of whole body lipolytic rate, was greater in women than in men (2.1 ± 0.2 vs. 1.5 ± 0.1 μmol · kg body wt⁻¹ · min⁻¹; P < 0.05). However, the relative increase in glycerol R_a with continued fasting was blunted in women compared with men (40 ± 7 vs. 80 ± 4% increase; P < 0.05), resulting in similar lipolytic rates in both genders at 22 h (2.8 ± 0.2 and 2.6 ± 0.1 μmol · kg body wt⁻¹ · min⁻¹ for women and men, respectively). In contrast, glucose R_a was similar in men and women at 14 h (11 ± 0.6 vs. 12 ± 0.7 μmol · kg body wt⁻¹ · min⁻¹) and 22 h of fasting (9 ± 0.6 vs. 10 ± 0.6 μmol · kg body wt⁻¹ · min⁻¹). These data demonstrate the presence of sexual dimorphism in lipid, but not glucose, metabolism during both basal and short-term fasting conditions.