The purpose of this study was to evaluate the effects of longitudinal drift in scanner hardware, inter-scanner variability (bias), and scanner upgrade on longitudinal changes in global and regional diffusion properties using longitudinal data obtained on two scanners of the exact same model at one institution. A total of 224 normal subjects were scanned twice, at an interval of about 1 year, using two 3.0-T scanners of the exact same model. Both scanners were simultaneously upgraded during the study period. The subjects were divided into four groups according to the combination of scanners used. With use of tract-based spatial statistics, we evaluated the effects of scanner drift and inter-scanner variability (bias) on global and regional fractional anisotropy (FA), axial diffusivity (AD), and radial diffusivity (RD) changes of the white matter. Even with scanners of the exact same model, inter-scanner variability (bias) significantly affected longitudinal results. FA, AD, and RD of the white matter were relatively stable within the same scanner. We also investigated the effect of scanner upgrade on longitudinal FA, AD, and RD changes. The scanner upgrade included only software upgrade, not hardware upgrade; however, there was a significant effect of scanner upgrade on longitudinal results. These results indicate that inter-scanner variability and scanner upgrade can significantly affect the results of longitudinal diffusion tensor imaging studies.