Office workers are vulnerable to the adverse health effects of sedentary behavior (i.e., sitting time). Increasing physical activity and preventing time spent sitting is an occupational health priority. This randomized crossover design study compared the short-term (3-days) effects of hourly interruptions of sedentary time with 5-min micrrobouts of activity for 9 hours (MICRO) to a sedentary control condition (SED) and a duration-matched continuous single bout of physical activity (45-min/d, ONE) condition on inclinometer-derived sitting-time on work and non-work days in sedentary overweight/obese adults. Differences in sitting/lying, standing, stepping, number of sit/stand transitions, time spent in moderate and vigorous activity (MVPA), energy expenditure, self-perceived vigor and fatigue, and insulin sensitivity were also examined. Twenty-two participants (10M/12F; 31.7 ± 1.3 year old BMI 30.4 ± 0.5 kg/m2) completed all conditions. No between-condition effects were observed in sitting-time and sit/stand transitions. Both interventions increased daily steps, MVPA and energy expenditure with increases being greater in ONE than MICRO. Feelings of vigor and fasting insulin sensitivity were also improved. Participants reported less fatigue with MICRO than SED and ONE. Both interventions increase physical activity and energy expenditure in occupational and leisure-time contexts. The sustainability of these effects over the long term and on health outcomes will need to be tested in future studies.