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<h4>Purpose</h4>End-stage renal disease (ESRD) patients exhibit silent white-matter alterations after long-term hemodialysis, which may be due to ESRD itself or the hemodialysis. The purpose of this study was to investigate the longitudinal white-matter alterations in the ESRD patients under 3-year long-term hemodialysis using voxel-wise analysis of diffusion tensor imaging (DTI).<h4>Materials and methods</h4>15 ESRD patients and 15 age-matched healthy controls participated in this study. Due to the development of abnormal brain lesions in some cases, 13 ESRD patients and 13 age-matched healthy controls were enrolled and underwent cognitive function assessment and DTI acquisition at two-time points separated by 3 years. Voxel-based analysis was performed to globally detect white-matter alterations between the two groups as well as between the two scans within the two groups.<h4>Results</h4>In the ESRD patients, diffusivity indices were significantly increased and the fractional anisotropy was significantly decreased in both scans, as compared with healthy controls. Longitudinal comparisons showed significant white-matter alterations in healthy controls in three years, but little or no significant alterations were noted in the ESRD patients after additional 3-year hemodialysis.<h4>Conclusion</h4>Poorer white matter integrity and cognitive function are noted in ESRD patients and the toxic effect of ESRD may be the major factor of white matter alterations.