Endothelial function and microvesicle concentration changes after acute bouts of continuous eccentric exercise have not been assessed previously nor compared with concentric exercise at similar aerobic power outputs. This method of training may be useful among some clinical populations, but acute responses are not well described. As such, 12 healthy males completed 2 experimental sessions of either 45 min of eccentric or concentric cycling at a matched aerobic power output below the ventilatory threshold. Brachial artery vascular function was assessed throughout 5 min of forearm ischemia and 3 min thereafter, before and at 5 and 40 min of recovery following each exercise session [flow-mediated dilation (FMD)]. Venous blood samples were acquired before each vascular function assessment. FMD significantly decreased after eccentric cycling by 40 min of recovery (P < 0.05), but was unaltered after concentric exercise. No differences in peak hyperemic blood flow velocity occurred neither between modalities nor at any time point (P > 0.05). Platelet-derived microvesicles increased by ~20% after both exercise modalities (P < 0.05) while endothelial-derived microvesicles were unchanged (P > 0.05). Moderate relationships with cardiac output, a surrogate for shear stress, and norepinephrine were apparent (P < 0.05), but there were no relationships with inflammatory or acute phase proteins. In summary, eccentric endurance exercise induced macrovascular endothelial dysfunction; however, endothelial activation determined by endothelial microvesicles did not occur suggesting that this modality may induce oxidative stress but no significant endothelial damage. In addition, the increase in platelet microvesicle concentrations may induce beneficial microvascular adaptations as suggested by previous research.NEW & NOTEWORTHY Continuous eccentric cycling exercise induces substantial skeletal muscle, tendon, and bone strain providing a potentially beneficial stimulus among clinical populations. This modality also induces temporary endothelial dysfunction but no apparent damage or activation of the endothelium indicated by microvesicle production, whereas proangiogenic platelet microvesicles are released similarly following both concentric and eccentric cycling and may relate to the shear stress and catecholamine response to exercise.