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Purpose Retinal Vessel Analyzer (RVA) is a validated instrument to measure retinal vessel diameter in humans. The purpose of this study was to assess the inter‐observer reproducibility and intra‐observer repeatability of RVA with a microscope‐mounted fundus camera to determine retinal vessel diameter in minipigs. Methods The fundus image of 5 minipigs was recorded in digital videocassette for approximately 5 min under stable systemic arterial pH, pCO2, and pO2. In order to evaluate the inter‐observer reproducibility, each one of two investigators used RVA (Imedos GmbH, Jena, Germany) to measure the diameter of the superior temporal retinal artery on 5 separate 30‐sec recordings from each minipig. In order to evaluate the intra‐observer repeatability, one investigator performed 5 measurements on a single randomly selected 30‐sec recording from each minipig. Inter‐observer reproducibility was evaluated using Student's t‐test for independent samples and Pearson's correlation. Intra‐observer repeatability was assessed using coefficient of variation. Results There were no statistically significant differences between the two investigators (p=0.347). The measurements performed by the two investigators were significantly correlated (r=0.999; p<0.001). Coefficient of variation was 0.2%, indicating very high intra‐observer repeatability. Conclusion The results of the present study indicate that retinal vessel diameter can be reliably determined in minipigs using RVA with a microscope‐mounted fundus camera. [ABSTRACT FROM AUTHOR]