This study was aimed at evaluation of autonomic dysfunction in patients with multiple sclerosis (MS) by means of time- and frequency-domain parameters of heart rate variability (HRV) and conventional cardiovascular tests (deep breathing (DB) and active orthostatic test (AOT)). The study group enrolled 32 patients with the relapsing-remitting MS (17 m, 15 f, aged 29 ± 4.9 years, disease duration 4.2 ± 2.7 years, EDSS scores less than 3.0 and 26 subjects in good health (HC, 15 m, 11 f, aged 30.1 ± 2.7 years). In the MS group, at rest the variability of heart rate was decreased in comparison to the HC group seen by time- (SDNN, RMSSD, pNN50, CV, p < 0.01) and frequency-domain (TP, HF, LF, p < 0.05) parameters, what was indicative of the general decrease of the autonomic neurogenic control of the heart rate, both sympathetic and parasympathetic. The functional tests (DB and AOT) showed reduced cardiovascular reactivity in the MS group. Additionally, the cardio-respiratory synchronization was impaired in the MS group at rest and DB. The severity of HRV deficit in the MS group correlated with the activity of MS. In conclusion, the comprehensive assessment of time- and frequency-domain HRV parameters studied with functional tests provides better insight to understanding autonomic dysfunction in subjects with relapsing-remitting MS.