Short-term exercise can enhance insulin action, but the effect may be negated by the opposing action of energy surplus. The purpose of this investigation was to test the hypothesis that a single exercise bout would increase insulin action, even when opposed by a concurrent energy surplus. After 2 days in energy balance without exercise, baseline glucose and insulin areas under the curve and the insulin sensitivity index (C-ISI) were measured during an oral glucose tolerance test in 9 healthy, habitually active subjects (6 males, 3 females). A state of relative insulin insensitivity was then induced by systematic overfeeding (OF) to generate a daily energy surplus of 768 ± 203 kcal/d for 3 days, and the oral glucose tolerance test was repeated. In the following 24 hours, the OF was increased ∼2-fold (+6284 ± 1669 kJ/d) and subjects performed a single bout of exercise (expenditure = 3063 ± 803 kJ) to maintain the same energy surplus (+3125 ± 933 kJ/d; OF and exercise) as OF. After OF, fasting insulin tended to be higher (+36%, P = .099), insulin AUC rose by 38% (P = .002), and C-ISI declined from 6.6 ± 3.1 to 4.6 ± 1.8 (P = .007) compared with baseline. After OF and exercise, fasting insulin remained elevated (+43% compared with baseline; P = .043) and C-ISI rose only slightly (4.6 ± 1.8 to 5.2 ± 2.3; P = .058), but insulin AUC declined by 20% (P = .048) compared with OF. A single exercise bout, opposed by a concurrent energy surplus, decreased the insulin response to a glucose challenge, but only partially restored the insulin AUC to baseline and had no impact on C-ISI or fasting insulin concentrations.