Background Data regarding the autonomic control of heart rate in multiple sclerosis (MS) patients depending on the disease duration are lacking in the literature. The goal of this study was to evaluate differences in heart rate variability (HRV) in patients with MS according to the duration of the disease. Methods The study included 39 patients (23 female and 16 male; median age 42 years, range 34–53 years) with relapsing-remitting MS (RRMS) in stable phase and 39 age- and sex-matched healthy controls. RRMS patients were divided into two groups: group 1 with 21 patients within 5 years and group 2 with 18 patients with >5 years from the diagnosis of MS. HRV analysis was done with 24-h Holter ECG. Results Patients with RRMS had a significantly lower overall HRV than controls: SDNN 91 ± 18 msec vs. 135 ± 24 msec, p <0.001. RRMS patients with lower duration of disease (Group 1) had all higher HRV parameters except LF/HF ratio compared with RRMS patients with >5 years from the diagnosis of MS (Group 2): SDNN 94 ± 24 vs. 88 ± 21 msec, p = 0.008; TP 2028 ± 1326 vs. 1683 ± 1017 ms2 , p = 0.006. Conclusions Results of the study suggested that the autonomic control of heart rate depends on the disease duration in RRMS patients. Longer disease duration led to progressive impairment of cardiac autonomic balance in MS patients.