Background: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, which often causes progressive neurological disability. In addition to motor and sensory dysfunction, cognitive decline and fatigue are frequent manifestations of the disease. Fatigue is probably the most common symptom, with up to 90% of MS individuals reporting fatigue at some point. Cognitive impairment affects about 50% of patients and may be present at all MS stages. The aim of this observational study was to evaluate changes in cognition, fatigue, and disability status in 300 relapsing-remitting MS (RRMS) patients, treated with subcutaneous (sc) interferon (IFN) beta-1a over 2 years. Methods: The study was designed as an observational, multicentre, prospective, single-arm, phase IV study carried out in 13 MS centres in the Czech Republic. Cognition status was assessed using the Paced Auditory Serial Addition Task (PASAT), fatigue using the Fatigue Descriptive Scale (FDS), and disability using the Expanded Disability Status Scale (EDSS), at baseline, and after 6, 12 and 24 months. The percentage of patients with changed versus stable cognition, fatigue status and disability was calculated at each time point and the changes in these scores were evaluated. Results: The proportion of patients with cognitive improvement was higher compared with those with a stable or decreased PASAT scores at all time points, and the average cognitive performance improved during the follow-up period. Also the proportion of patients with stable or improved fatigue and EDSS scores was higher compared with those in which FDS or EDSS scores declined, this was found at all time points of the analysed sample. However, the direct effect of IFN beta-1a on cognition and fatigue cannot be concluded from this study. Conclusions: The results of this observational study have demonstrated a stable or improved cognitive performance, fatigue status, and disability level in the majority of RRMS patients treated with sc IFN beta-1a over a two-year follow-up period, in a real life setting, in the Czech Republic.