To investigate the repeatability and reproducibility of manual measurements of the reflectivity of retinal layers in healthy eyes and in eyes with acute retinal vessel occlusion via spectral-domain optical coherence tomography (SD-OCT). To reflect the full spectrum of different reflectivities seen in OCT images, for each of the following conditions, exactly one patient with a healthy and an affected eye was selected: non-ischemic central retinal vein occlusion (CRVO), ischemic CRVO, incomplete, subtotal and total central retinal artery occlusion (CRAO). For all the patients, OCT images of the healthy and affected eyes were used. Each image was presented 10 times in a randomly changing sequence to all examiners (one experienced, two nonexperienced). The obtained values were analyzed via two statistical methods: Gage Repeatability & Reproducibility (Gage R&R, a measurement system analysis) and Lin's concordance correlation coefficient (Lin's CCC). The total gage R&R values were as follows: vitreous body (VB) 1.11%, innermost retinal layer (IMRL) 0.94%, middle retinal layer (MRL) 4.94%, inner retinal layer (IRL) 2.56% and outer retinal layer (ORL) 3.77%. The repeatability values were 0.79, 0.62, 2.80, 1.42 And 2.60, respectively. The reproducibility values were 0.32, 0.33, 2.41, 1.14 and 1.16, respectively. Our previously described method of manual measurement of SD-OCT images of retinal layers determining the level of reflectivity (and hence ischemia) in the retina without any invasive examination is reliable, repeatable, reproducible and feasible for clinical use. Registry: ClinicalTrials.gov, Protocol ID: EK 417,102,016, TRN: NCT03061526, Registration date: 19 February 2017.