Prolonged sitting is increasingly common and may possibly be unfavorable for cognitive function and mood. In this randomized crossover study, the effects of frequent, short physical activity breaks during prolonged sitting on cognitive task-related activation of the prefrontal cortex were investigated. The effects on working memory, psychological factors, and blood glucose were also examined, and whether arterial stiffness moderated prefrontal cortex activation. Thirteen subjects (mean age 50.5 years; eight men) underwent three 3-h sitting conditions, interrupted every 30-min by a different 3-min break on separate, randomized-ordered days: seated social interactions (SOCIAL), walking (WALK), or simple resistance activities (SRA). Arterial stiffness was assessed at baseline. Before and after each 3-h condition, psychological factors (stress, mood, sleepiness, and alertness) were assessed through questionnaires and functional near-infrared spectroscopy (fNIRS) was used to measure changes in prefrontal oxygenated hemoglobin (Oxy-Hb), indicative of cortical activation, while performing working memory tasks [1- (baseline), 2-, and 3-back]. Blood glucose levels were continuously measured throughout the conditions. Results revealed no significant changes in Oxy-Hb during the 2-back compared with the 1-back test in any condition, and no time-by-condition interactions. During the 3-back test, there was a significant decrease in Oxy-Hb compared with the 1-back after the WALK condition in the right prefrontal cortex, but there were no time-by-condition interactions, although 3-back reaction time improved only in the WALK condition. Mood and alertness improved after the WALK condition, which was significantly different from the SOCIAL condition. Arterial stiffness moderated the effects, such that changes in Oxy-Hb were significantly different between WALK and SOCIAL conditions only among those with low arterial stiffness. Blood glucose during the interventions did not differ between conditions. Thus, breaking up prolonged sitting with frequent, short physical activity breaks may reduce right prefrontal cortex activation, with improvements in some aspects of working memory, mood, and alertness. Clinical Trial Registration:www.ClinicalTrials.gov, identifier NCT04137211.