Purpose: To investigate diurnal changes of choroidal sublayer perfusion in normal eyes and to identify influencing factors using optical coherence tomography angiography (OCTA). Methods: A prospective study was conducted on healthy volunteers, each of whom underwent repeated measurements of subfoveal choroidal thickness (SFCT) via enhanced depth imaging (EDI) optical coherence tomography (OCT) as well as perfusion of choroidal vascular sublayers using OCTA at 7 a.m., 12 p.m., 4 p.m. and 8 p.m. Possible interactions between diurnal variations and other factors, such as mean arterial pressure (MAP), gender and age, were evaluated. Results: A total of 22 eyes from 22 participants were analysed. Mean age of participants was 56 years. A significant pattern of diurnal variation was observed for SFCT (p < 0.001) as well as perfusion of Sattler's layer (SLP; p = 0.009) and Haller's layer (HLP; p = 0.003). SFCT demonstrated a linear decrease, being thicker in the morning (348 lm) and thinner in the evening (310 lm). Both, SLP and HLP showed a quadratic relation to time of day, increasing from morning (64% and 76%) to afternoon (66% and 77%), before decreasing again in the evening (64% and 76%). HLP changes were significantly associated with fluctuations of MAP (B = 0.0007; CI 0.0001-0.0014; p = 0.047). No significant differences with regard to gender were detectable. However, older participants (≥60 years) had fewer diurnal changes (p = 0.042). Conclusion: Optical coherence tomography angiography-based analysis of choroidal sublayer perfusion demonstrated significant diurnal variations. Therefore, it is important to account for time of day, when comparing longitudinal OCTA data.