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To investigate associations of central retinal arteriolar equivalent (CRAE), a measure of retinal arteriolar width, and central retinal venular equivalents (CRVE), a measure of retinal venular width, with shiftwork in 199 police officers (72.9% men).Shiftwork (day, afternoon, night) was assessed using electronic payroll records. Four digital retinal images per officer were taken. Mean diameters of the retinal vasculature were compared across shifts using analysis of variance (ANOVA)/analysis of covariance (ANCOVA).Among all officers (mean age = 46.6 ± 6.8 years), shiftwork was not significantly associated with CRAE or CRVE. However, among current and former smokers, night-shift officers had a wider mean (±standard error [SE]) CRVE (230.0 ± 4.5 μm) compared with day shift officers (215.1 ± 3.5 μm); adjusted P = 0.014.Night shift schedule in current and former smokers is associated with wider retinal venules. Reasons for this association are not known. Longitudinal studies are warranted.