PURPOSE: To identify the investigator effect in the analysis results of Heidelberg retinal flowmetry (HRF) images when pixel-by-pixel analysis is performed.METHODS: Thirty-two of 732 HRF images were randomly selected from a population-based study. Pixel-by-pixel analysis was performed by two trained masked graders in the following way: a square window of 40 x 40 pixels or two windows of 30 x 30 pixels or four windows of 20 x 20 pixels free from blood vessels at the peripapillary retina were identified. Using a 1 x 1-pixel window, the grader performed pointwise analysis according to a specific protocol. The analysis process was performed by each observer three times (A, B, C) at 1-week intervals. The percentage of pixels with < 1 arbitrary unit of flow (zero flow), the 10th, 25th, 50th, 75th and 90th percentiles and mean flow values were calculated. The difference between the results of analyses B-A and C-A for all HRF parameters was estimated using the Wilcoxon signed rank test. Mixed-effect regression models were also used after controlling for grader effect and correlation within subjects.RESULTS: There was no statistically significant difference between the results of analyses B-A and C-A or for any parameter in the mixed-effect regression models. Intraclass correlation was 0.9665 for the percentage of zero flow pixels.CONCLUSIONS: Pixel-by-pixel analysis of HRF images by trained graders remains a highly reproducible method. No grader effect was found. If a precise protocol is followed, the results are independent of the exact placement of the analysis windows and the pointwise analysis of the identified and mapped retinal tissue.