Periods of prolonged sitting impairs endothelial function in lower limb conduit arteries, which is attenuated with physical activity breaks. The effect of activity breaks on upper limb arteries has not been examined. This study assessed changes in brachial artery endothelial function following either a prolonged sitting period or breaking up this sedentary time by performing sets of callisthenics exercises. Ten healthy participants (6 men) completed 2 conditions in a counterbalanced order: (a) 1-h 26-min sitting, or (b) breaking up this period every 20 min by performing a set of 5 callisthenics exercises. Brachial artery endothelial function was assessed via ultrasound using the flow-mediated dilation (FMD) technique prior to and following each condition, while brachial shear rate (SR) was acquired after each set of callisthenics. There was no significant change in FMD over time (P = 0.09) or between conditions (P = 0.12). Compared to sitting, brachial SR increased following each set of callisthenics, with a significant difference after the third break (Sit: 33.94 ± 12.79 s-1; Callisthenics: 57.16 ± 30.48 s-1, P = 0.02). Alterations in SR in the upper limbs suggest callisthenics may be an effective intervention to break up sedentary time and attenuate the potentially deleterious effects of prolonged sitting on cardiovascular health.