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To measure the retinal arteriole and venule oxygen saturation (SO2) using a conventional fundus camera, retinal oximetry based on nonsimultaneous image acquisition was developed and evaluated. Two retinal images were sequentially acquired using a conventional fundus camera with two bandpass filters (568 nm: isobestic, 600 nm: nonisobestic wavelength), one after another, instead of a built-in green filter. The images were registered to compensate for the differences caused by eye movements during the image acquisition. Retinal SO2 was measured using two wavelength oximetry. To evaluate sensitivity of the proposed method, SO2 in the arterioles and venules before and after inhalation of 100% O2 were compared, respectively, in 11 healthy subjects. After inhalation of 100% O2, SO2 increased from 96.0 ± 6.0% to 98.8% ± 7.1% in the arterioles (p = 0.002) and from 54.0 ± 8.0% to 66.7% ± 7.2% in the venules (p = 0.005) (paired t-test, n = 11). Reproducibility of the method was 2.6% and 5.2% in the arterioles and venules, respectively (average standard deviation of five measurements, n = 11).