A novel clinical methodology is proposed, which characterizes in-vivo arterial stiffness in the central microcirculation. Pulse wave propagation in retinal arteries is assessed and a parameter “retinal pulse-wave velocity" (rPWV) is calculated. We demonstrated previously that both aging with not excluded cardiovascular risk factors and mild arterial hypertension are associated with elevated rPWV. Whether rPWV increases with age in a cohort of medically validated healthy subjects is investigated. 71 healthy 41.0±12.1 (range: 20 – 66) years old volunteers were examined. The following cardiovascular risk factors were excluded: overweight, increased blood pressure, cholesterol level and blood glucose. Time dependent alterations of vessel diameter were assessed by the Dynamic Vessel Analyzer in a segment of a retinal artery. The data was filtered and evaluated by methods of mathematical signal analysis in order to obtain rPWV value. rPWV amounted to 370±100 (range: 180 – 620) units/second in the whole group (units correspond to μm in the Gullstrand’s eye model). In the cohort rPWV increased with age (Pearson’s correlation: r=0.41, p<0.005) to 21 units/second per a decade. rPWV showed weak correlations with vessel diameter: r=−0.27, p<0.05 and mean arterial pressure: r=0.22, p<0.05. Thus healthy aging with excluded mentioned cardiovascular risk factors is associated with a flat increase of rPWV and hence with age-related elevation of retinal arterial stiffness. This rPWV alteration and absolute rPWV values in medically validated volunteers allow to distinguish this cohort well from young hypertensives (1620±1310 units/second) or aged subjects with not excluded cardiovascular risk (1200±520 units/second) reported in our previous studies.