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Background and purpose: This study employs magnetic resonance imaging (MRI) diffusion tensor imaging to compare diffusion measures in the brains of patients with Parkinson's disease (PD) with healthy controls using longitudinal data. Methods: One-hundred and twenty-two patients and 34 controls were included at baseline. The MRI investigations were repeated after 1, 3 and 5 years. The diffusion measures were quantified using fractional anisotropy and mean, radial and axial diffusion (FA, MD, RD, AD). Regions of interest included the anterior, middle and posterior substantia nigra (SN), but also other areas. Linear models were used to test for the effect of disease and hemispheric lateralization. The P value was set at 0.05 (Bonferroni corrected). Results: Fractional anisotropy and AD were increased in the three nigral subareas in PD (P < 0.01), but MD and RD were unaltered. The right SN had higher FA than the left in all subareas (P < 0.01). MD and AD were increased in the right anterior part (P < 0.04), whereas MD and RD were decreased in the right middle and posterior parts (P < 0.001). The left middle cerebellar peduncle had increased FA and AD (P < 0.001) and decreased MD and RD (P < 0.01) compared to the right. Diffusion measures did not progress over time and side differences were not related to disease or lateralization of symptoms. Conclusions: Increased FA in the SN in PD indicates gliosis and inflammation in the nuclei, but possibly also intrusion of surrounding fibres into the shrinking structure. The hemispheric side differences of diffusion might reflect natural lateralization of connectivity, but their relation to PD must be studied further.