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Retinal oxygen saturation (SO2) during flicker light stimulation was measured non-invasively in humans with age-related macular degeneration (AMD). Furthermore, the differences between early and late stages of AMD were evaluated. In 60 eyes of 45 AMD patients (74 ± 8.3 years) and 23 eyes of 23 healthy controls (73.4 ± 7.4 years), the SO2 of arterioles and venules was measured with the oximetry module of the Retinal Vessel Analyzer. Arterial SO2, venous SO2 and arteriovenous SO2 difference at baseline and with the flicker were assessed and compared with controls. From the difference between the arteriovenous SO2 under flicker stimulation and at baseline, the parameter delta av. Diff was calculated. Subgroup analyses of non-exudative (dry) AMD, exudative (wet) AMD and their end stages, geographic atrophy (GA) and disciform scar (DS) were performed. In comparison with healthy subjects (mean − 4.90, CI [− 6.32, − 3.43]), the parameter delta av. Diff was significantly reduced in all AMD patients (mean − 2.20, CI − 3.15, −1.23, p = 0.003), dry AMD (mean − 1.97, CI − 3.31, −0.63, p = 0.013) and wet AMD (mean − 2.35, CI − 3.50, − 1.19, p = 0.025). The comparison between wet and dry AMD revealed no significant results (p = 1). The comparison between AMD subgroups and healthy controls (median (IQR) − 4.29 (− 8.32; − 2.42) %) showed significant differences in non-neovascular (early dry AMD) (median (IQR) − 2.43 (− 4.59; − 0.74) %, p = 0.038), GA (median (IQR) 0.10 (− 4.02; 3.15) %, p = 0.019) and DS (median (IQR) − 1.67 (− 3.52; − 0.12) %, p = 0.03). A nearly significant reduction was observed in exudative (early wet) AMD (median (IQR) − 2.71 (− 5.84; − 0.2) %, p = 0.055). Minimal, not statistically significant differences of delta av. Diff were found between AMD subgroups. None of the baseline parameters was significantly different between patients and healthy controls, even after flicker light stimulation. Non-invasive retinal oximetry with flicker light stimulation seems to be a suitable method to study the pathogenetic mechanisms of AMD. The mathematically derived parameter delta av. Diff appears to be more sensitive than arteriovenous SO2 difference. Results suggest that the regulation of retinal oxygen supply, oxygen consumption or both is impaired in AMD.