BACKGROUND: While physical activity has been shown to improve cognitive performance and well-being, office workers are essentially sedentary. We compared the effects of physical activity performed as (i) one bout in the morning or (ii) as microbouts spread out across the day to (iii) a day spent sitting, on mood and energy levels and cognitive function. METHODS: In a randomized crossover trial, 30 sedentary adults completed each of three conditions: 6 h of uninterrupted sitting (SIT), SIT plus 30 min of moderate-intensity treadmill walking in the morning (ONE), and SIT plus six hourly 5-min microbouts of moderate-intensity treadmill walking (MICRO). Self-perceived energy, mood, and appetite were assessed with visual analog scales. Vigor and fatigue were assessed with the Profile of Mood State questionnaire. Cognitive function was measured using a flanker task and the Comprehensive Trail Making Test. Intervention effects were tested using linear mixed models. RESULTS: Both ONE and MICRO increased self-perceived energy and vigor compared to SIT (p < 0.05 for all). MICRO, but not ONE, improved mood, decreased levels of fatigue and reduced food cravings at the end of the day compared to SIT (p < 0.05 for all). Cognitive function was not significantly affected by condition. CONCLUSIONS: In addition to the beneficial impact of physical activity on levels of energy and vigor, spreading out physical activity throughout the day improved mood, decreased feelings of fatigue and affected appetite. Introducing short bouts of activity during the workday of sedentary office workers is a promising approach to improve overall well-being at work without negatively impacting cognitive performance. TRIAL REGISTRATION: NCT02717377 , registered 22 March 2016.