In this study, we tested the hypothesis that breaking up prolonged sitting with isometric exercise would be a useful sitting break strategy to promote cardiovascular health and cognitive benefits in healthy adults. This is a three-condition randomized crossover study on which participants performed, in randomized order, the following 3-hour conditions: Sitting with no breaks (Control); Sitting with 2 min isometric leg extension exercise breaks every 30 min (Isometric); Sitting with 2 min light-walking exercise breaks every 30 min. Cardiovascular parameters (i.e. Vascular function, blood pressure, cardiac autonomic modulation) and cognition (Strrop and Eriksen-Flanker tests) were measured before, post 10 min and post 50 min. Our findings did not indicate any significant effects of breaking up sitting with isometric exercise in any of the cardiovascular parameters measured, showing that isometric exercise breaks do not seem to lead to significant effects on cardiovascular health.