Hypertension is characterized by microvascular remodeling resulting in an increased wall/lumen ratio. Increased microvascular stiffness contributes to an increase in wall/lumen ratio. We aimed to investigate the possibility to transform the measurement of macrovascular stiffness into a microvascular environment. We assessed retinal pulse wave velocity (rPWV) non-invasively in 65 male normoalbuminuric normotensive to mildly hypertensive subjects (age: 28.7±6.0 years). Time dependent alterations of retinal arterial diameter were measured by the Dynamic Vessel Analyzer. The data was filtered and evaluated by methods of signal analysis and rPWV was computed using three different methods. ‘Method1’ used filtration at heart rate (HR), ‘Method2’ filtered at higher HR multiples and ‘Method3’ used additionally linear fit for data averaging. Besides, office blood pressure (BP) and urinary albumine/creatinine ratio were assessed. ‘Method1’ was not associated with BP, while both methods applying filtration at high HR multiples showed a strong association with systolic BP throughout the cohort (r=0.49, r=0.63 P<0.001). Based on the highest association, ‘Method3’ was proposed to characterize rPWV. As the cohort was divided according to BP, mildly hypertensive patients showed significantly higher rPWV (1243±694 units/second) than subjects with high-normal BP (786±486 units/second, P<0.01) or normotensive subjects (442±148 units/second, P<0.001). Applying methodological principles for aortic PWV we consider rPWV as a non-invasive measure of microvascular stiffness. Our data suggests that filtration at higher HR multiples and linear fit result in strong association with BP. As our study was performed in normoalbuminuric subjects, rPWV may add detailed insights to early microvascular pathophysiology, potentially beyond microalbuminuria.