BACKGROUND AND OBJECTIVE: The Retinal Function Imager (RFI) (Optical Imaging Ltd., Rehovot, Israel) measures retinal blood flow velocity non-invasively. The authors studied the reproducibility of these measurements and assessed the effect of physiological components on them. PATIENTS AND METHODS: Sixty-seven individuals with no retinal pathology were recruited. Velocity reproducibility was verified by comparing repeated RFI measurements. The correlation of the velocity with physiological parameters was assessed by mixed linear and Gaussian models. RESULTS: The average velocity was 4.2 ± 0.9 mm/sec arterial and 3.3 ± 0.8 mm/sec venous. Variability was 7.5% ± 3.7% and interclass correlation coefficient was r = 0.744. Venous velocity decreased after 40 years of age (0.32 mm/sec per decade, P <.01). Arterial velocity increased as mean arterial pressure increased (0.25 mm/sec per 10 mm Hg, P <.01). There was also a positive association between velocities and heart rate (arteries: 0.21 mm/sec per 10 bpm, P <.05; veins: 0.22 mm/sec per 10 bpm, P <.01). CONCLUSION: The RFI provides a reproducible, noninvasive technique to assess retinal velocities.