Aortic stiffness is associated with augmented pressure pulsatility in large conduit arteries and remodeling of the microcirculation. However, studies in humans examining the relation between aortic stiffness and end-organ microvascular flow pulsatility are limited. Therefore, we used the retinal microvasculature as an end-organ in vivo model to examine the hypothesis that aortic stiffness would be positively associated with microvascular flow pulsatility index (PI) (flow pulse amplitude/mean flow) in humans. In 40 young/middle-age healthy adults (25-60 yr old, 50% women), aortic stiffness (carotid-femoral pulse wave velocity, CFPWV) and retinal arteriole flow (laser speckle flowgraphy) were examined at rest and during metabolic vasodilation (light flicker). CFPWV and related increases in central pulse pressure (PP) were inversely correlated with arteriole lumen diameter independent of age (CFPWV: R = -0.52, P = 0.001; Central PP: R = -0.39, P = 0.014). Accordingly, microvascular resistance was positively related to CFPWV independent of age (R = 0.35, P = 0.031). Multiple linear regression showed that CFPWV was not a significant determinant of resting arteriole flow PI (β = -0.10, P = 0.64). However, during reduced retinal microvascular resistance using light flicker (P < 0.001), CFPWV was a significant determinant of the percent change in arteriole flow PI (β = 0.58, P = 0.046), but not mean flow (β = -0.17, P = 0.54), where reductions in arteriole flow PI were associated with lower CFPWV. In summary, our findings suggest that higher aortic stiffness and the related increase in central PP in healthy young/middle-age adults are associated with retinal arteriole narrowing and smaller reductions in arteriole flow pulsatility in response to dynamic conditions such as local metabolic vasodilation.NEW & NOTEWORTHY By using the human retinal microvasculature as an end-organ in vivo model, we confirm that aortic stiffness and related increases in central pulse pressure are inversely correlated with retinal arteriole lumen diameter and increased microvascular resistance among heathy young/middle-age adults. Additionally, higher aortic stiffness is not associated with excessive flow pulsatility in the retinal microvasculature under tonic conditions but may be related to limited reductions in retinal arteriole flow pulsatility in response to local vasodilation.