To assess the reproducibility of measurement of retinal nerve fiber layer (RNFL) thickness among subjects using the GDx Nerve Fiber Analyzer (NFA). One eye of each of five young and healthy volunteers was imaged by three newly trained technicians (R.-H.P, K.-R.K., and S.-T.K.). Each volunteer was scanned six consecutive times for five independent times within a 1-month period. One composite image was selected out of three best images per session. Intra- and interoperator reproducibilities were calculated and the influence of placement of measurement ellipse by a single operator on interobserver reproducibility was also investigated. Mean coefficients of variation for total integral value of RNFL were 3.67 +/- 1.47%, 3.86 +/- 2.13%, and 4.16 +/- 1.97%, respectively for the three technicians. Interoperator variability was not statistically significant (P = 0.075), and the difference was even less if a single ellipse drawn by one operator was used in all measurements (P = 0.1528) by repeated-measures analysis of variance (ANOVA). The results suggest that GDx NFA may provide acceptable intraoperator as well as interoperator reproducibility among Asian eyes.