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Fatigue is one of the most disabling symptoms in multiple sclerosis (MS) patients. There is no or only weak correlation between conventional magnetic resonance imaging (MRI) parameters and level of fatigue. The aim of this study was to investigate the relationship between progression of corpus callosum (CC) atrophy and fatigue in MS patients. This was a cohort study in 70 patients with relapsing form of MS (RRMS) and serial MRIs over a mean follow-up of 4.8 years [67% female, mean age 42 ± 11 years, mean disease duration 9.7 ± 7.6 years, mean Expanded Disability Status Scale (EDSS) 2.8 ± 1.6]. Fatigue was assessed by the Fatigue Severity Scale (FSS). CC size was measured with the CC index (CCI). In total, 40% of the patients suffered from fatigue (mean FSS score 5.3 ± 1.1) and 60% patients had no fatigue (mean FSS score of 2.1 ± 1). Patients with fatigue had higher EDSS scores (p = 0.01) and CC atrophy was more pronounced in patients with fatigue (-21.8 vs. -12.1%, p = 0.005). FSS correlated with CCI change over time (r = -0.33; p = 0.009) and EDSS (p = 0.008; r = 0.361). The association between annualized CCI change and FSS was independent from EDSS, disease duration, gender and age in a multivariate linear regression analysis (p < 0.001). Progression of CC atrophy may play a role in the evolution of MS-related fatigue.