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Retinal vessel phenotype is predictive for cardiovascular outcome. This cross-sectional population-based study aimed to quantify normative data and standard operating procedures for static and dynamic retinal vessel analysis. We analysed central retinal arteriolar (CRAE) and venular (CRVE) diameter equivalents, as well as retinal endothelial function, measured by flicker light‐induced maximal arteriolar (aFID) and venular (vFID) dilatation. Measurements were performed in 277 healthy individuals aged 20 to 82 years of the COmPLETE study. The mean range from the youngest compared to the oldest decade was 196 ± 13 to 166 ± 17 µm for CRAE, 220 ± 15 to 199 ± 16 µm for CRVE, 3.74 ± 2.17 to 3.79 ± 2.43% for aFID and 4.64 ± 1.85 to 3.86 ± 1.56% for vFID. Lower CRAE [estimate (95% CI): − 0.52 (− 0.61 to − 0.43)], CRVE [− 0.33 (− 0.43 to − 0.24)] and vFID [− 0.01 (− 0.26 to − 0.00)], but not aFID, were significantly associated with older age. Interestingly, higher blood pressure was associated with narrower CRAE [− 0.82 (− 1.00 to − 0.63)] but higher aFID [0.05 (0.03 to 0.07)]. Likewise, narrower CRAE were associated with a higher predicted aFID [− 0.02 (− 0.37 to − 0.01)]. We recommend use of defined standardized operating procedures and cardiovascular risk stratification based on normative data to allow for clinical implementation of retinal vessel analysis in a personalized medicine approach.