The aim of this study was to determine whether a bout of morning exercise (EXE₁) can alter neuroendocrine and metabolic responses to subsequent afternoon exercise (EXE₂) and whether these changes follow a gender-specific pattern. Sixteen healthy volunteers (8 men and 8 women, age 27 ± 1 yr, body mass index 23 ± 1 kg/m², maximal O₂ uptake 31 ± 2 ml · kg¹ · min¹) were studied after an overnight fast. EXE₁ and EXE₂ each consisted of 90 min of cycling on a stationary bike at 48 ± 2% of maximal O₂ uptake separated by 3 h. To avoid the confounding effects of hypoglycemia and glycogen depletion, carbohydrate (1.5 g/kg body wt po) was given after EXE₁, and plasma glucose was maintained at euglycemia during both episodes of exercise by a modification of the glucose-clamp technique. Basal insulin levels (7 ± 1 µU/ml) and exercise-induced insulin decreases (3 µU/ml) were similar during EXE₁ and EXE₂. Plasma glucose was 5.2 ± 0.1 and 5.2 ± 0.1 mmol/l during EXE₁ and EXE₂, respectively. The glucose infusion rate needed to maintain euglycemia during the last 30 min of exercise was increased during EXE₂ compared with EXE₁ (32 ± 4 vs. 7 ± 2 µmol · kg¹ · min¹). Although this increased need for exogenous glucose was similar in men and women, gender differences in counterregulatory responses were significant. Compared with EXE₁, epinephrine, norepinephrine, growth hormone, pancreatic polypeptide, and cortisol responses were blunted during EXE₂ in men, but neuroendocrine responses were preserved or increased in women. In summary, morning exercise significantly impaired the body's ability to maintain euglycemia during later exercise of similar intensity and duration. We conclude that antecedent exercise can significantly modify, in a gender-specific fashion, metabolic and neuroendocrine responses to subsequent exercise.