Although regular exercise is associated with reduced cardiometabolic disease risk among overweight adults, it remains unclear whether much of the health benefits of exercise are derived from the most recent session(s) of exercise or if they are the result of adaptations stemming from weeks, months, or even years of training. The purpose of this study was to compare the effects of habitual and acute exercise on key markers of cardiometabolic disease risk in overweight adults. We compared insulin sensitivity index (ISI) using an oral glucose tolerance test, blood pressure (BP), blood lipids, and systemic inflammatory cytokines in 12 overweight to mildly obese adults (BMI: 27-34 kg/m(2)) who exercise regularly (EX; >2.5 h exercise per week) with a well-matched cohort of 12 nonexercisers (Non-EX). Baseline measurements in EX were performed exactly 3 days after exercise, whereas Non-EX remained sedentary. We repeated these measurements the day after a session of exercise in both groups. At baseline, ISI was significantly greater in EX versus Non-EX (3.1 ± 0.2 vs. 2.3 ± 0.2; p = 0.02), but BP, blood lipids, and plasma concentration of the systemic inflammatory cytokines we measured were not different between groups. Acute exercise increased ISI the next morning in Non-EX (2.3 ± 0.2 vs. 2.8 ± 0.3; p = 0.03) but not EX. As a result, ISI was similar between groups the morning after exercise. In summary, exercising regularly was accompanied by a persistent improvement in insulin sensitivity that lasted at least 3 days after exercise in overweight adults, but just one session of exercise increased insulin sensitivity among sedentary overweight adults to levels equivalent to the regular exercisers.