PURPOSE: To evaluate the long-term variability of GDx VCC retinal nerve fiber layer (RNFL) thickness measurements. METHODS: The study enrolled a cohort of glaucoma suspects who did not develop any evidence of visual field damage or change in the appearance of the optic nerve during an average follow-up of 9.1+/-3.2 years. Subjects underwent ocular imaging using the commercially available GDx VCC scanning laser polarimeter. At each visit, each eye was imaged 3 times. Subjects underwent repeated testing with GDx VCC at approximately 12-month intervals during their follow-up. In total, 255 examinations were obtained in 31 eyes of 31 individuals during an average GDx VCC follow-up time of 26.0+/-8.9 months. A random effects analysis of variance model was used to estimate intraclass correlation coefficients and long-term and short-term variability estimates. RESULTS: Intraclass correlation coefficients ranged from 0.77 to 0.86 for GDx VCC parameters. Short-term variability estimates ranged from 2.45 to 3.89 microm for RNFL thickness parameters, whereas the short-term variability estimate for the parameter Nerve Fiber Indicator was 3.71. Long-term variability was slightly higher than short-term variability for all parameters. For RNFL thickness parameters, long-term variability estimates ranged from 3.21 to 4.97 microm, whereas for the parameter Nerve Fiber Indicator the long-term variability estimate was 4.93. CONCLUSIONS: RNFL measurements obtained with the GDx VCC were found to be highly reproducible in a long-term test-retest situation, supporting the use of this instrument for longitudinal assessment of the RNFL.