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The purpose of our study was to investigate the potential pitfalls associated with different vessel segmentation methods using the built-in software of the Retinal Function Imager (RFI) for the analysis of retinal blood flow velocity (BFV). Ten eyes of nine healthy subjects were enrolled in the study. Retinal blood flow measurements were obtained with the RFI device with a 20° field of view imaging. The same grader segmented the retinal vasculature using the RFI software in both sessions, with segments ranging in length from 50 to 100pixels ("short segments