Optical coherence tomography angiography measurements are influenced by a range of environmental factors as blood pressure and physical fitness. The present study aimed to evaluate the effects of light and dark exposure in eyes with neutral and mydriatic pupils on vessel density in the macular and optic nerve head regions, as measured using optical coherence tomography angiography (OCTA). 55 eyes of 55 healthy volunteers (28 patients with neutral pupils; 27.18 ± 4.33 years) were examined using a high-speed and high-resolution spectral-domain OCT XR Avanti system with a split-spectrum amplitude de-correlation angiography algorithm. OCTA imaging was performed after dark adaptation and after exposure to light. The vessel density data of the superficial and deep retinal macular and optic nerve head region OCT-angiogram were analyzed for these two light conditions. Through Bonferroni correction for multiple testing, the p- value was adapted from 0.05 to 0.017. In eyes with neutral pupils, a significant increase was found in the capillary region of the optic nerve head region (p = 0.002), comparing dark- and light-adaptation. In the macular region of eyes with neutral (p = 0.718) and mydriatic pupils (p = 0.043), no significant differences were observed, as were any in the optic nerve head region of the mydriatic eyes (p = 0.797). This observation suggests that light conditions could be a possible factor influencing OCTA measurements. After dark exposure, vessel density data were significantly different between eyes with neutral and mydriatic pupils (nerve head region: p < 0.0001, superficial macula: p < 0.0001, deep macula: p = 0.0025). These data warn for the effect of mydriatic drops on vessel density measurements.