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Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a novel modality to investigate the human retina. This study aims to characterize the effects of age, pigmentation, and gender in FLIO. A total of 97 eyes from 97 healthy subjects (mean age 37 ± 18 years, range 9-85 years) were investigated in this study. This study included 47 (49%) females and 50 males. The pigmentation analysis was a substudy including 64 subjects aged 18 to 40 years (mean age 29 ± 6 years). These were categorized in groups A (darkly pigmented, 8), B (medium pigm