Fitness and exercise may counteract the detrimental metabolic and mood adaptations during prolonged sitting. This study distinguishes the immediate effects of a single bout vs. work-load and intensity-matched repeated exercise breaks on subjective well-being, blood glucose, and insulin response (analyzed as area under the curve) during sedentary time; and assesses the influence of fitness and caloric intake on metabolic alterations during sedentariness. Eighteen women underwent cardiopulmonary exercise testing and three 4 h sitting interventions: two exercise interventions (70% VO2max, 30 min, cycle ergometer: (1) cycling prior to sitting; (2) sitting interrupted by 5 × 6 min cycling), and one control condition (sitting). Participants consumed one meal with ad libitum quantity (caloric intake), but standardized macronutrient proportion. Exercise breaks (4057 ± 2079 μU/mL·min) reduced insulin values compared to a single bout of exercise (5346 ± 5000 μU/mL·min) and the control condition (6037 ± 3571 μU/mL·min) (p ≤ 0.05). ANCOVA revealed moderating effects of caloric intake (519 ± 211 kilocalories) (p ≤ 0.01), but no effects of cardiorespiratory fitness (41.3 ± 4.2 mL/kg/min). Breaks also led to lower depression, but higher arousal compared to a no exercise control (p ≤ 0.05). Both exercise trials led to decreased agitation (p ≤ 0.05). Exercise prior to sitting led to greater peace of mind during sedentary behavior (p ≤ 0.05). Just being fit or exercising prior to sedentary behavior are not feasible to cope with acute detrimental metabolic changes during sedentary behavior. Exercise breaks reduce the insulin response to a meal. Despite their vigorous intensity, breaks are perceived as positive stimulus. Detrimental metabolic changes during sedentary time could also be minimized by limiting caloric intake.