Purpose: To evaluate changes in the retinal microvasculature of young adults over 24 hours using optical coherence tomography angiography (OCT-A). Methods: Participants (n = 44, mean age 23.2 ± 4.1 years, 24 myopes and 20 nonmyopes) with normal ophthalmological findings were recruited. Two macular OCT-A and OCT scans, systemic blood pressure, intraocular pressure (IOP), and biometry measurements were taken every four hours over 24 hours. Superficial and deep retinal layer en face images were analyzed to extract magnification-corrected vascular indexes using image analysis including foveal avascular zone metrics, vessel density, and perfusion density for the foveal, parafoveal, and perifoveal zones. Results: Significant diurnal variations (P < 0.001) were observed in the vessel and perfusion density in the three superficial retinal layer regions, with acrophase between 4:30 PM and 8:30 PM. Only foveal and parafoveal regions of the deep retinal layer exhibited significant diurnal variations with acrophase between 9 AM and 3 PM. Myopes and nonmyopes had different acrophases but not amplitudes in the parafoveal perfusion density of superficial retinal layer (P = 0.039). Significant correlations were observed between diurnal amplitudes or acrophases of superficial retinal layer indexes and systemic pulse pressure, IOP, axial length and retinal thickness. Conclusions: This study shows, for the first time, that significant diurnal variation exists in OCT-A indexes of macular superficial and deep retinal layer over 24 hours and were related to variations in various ocular and systemic measurements. Myopes and nonmyopes showed differences in the timing but not in amplitude of the superficial retinal layer parafoveal perfusion density variations but not in deep retinal layer.