Little is known about the interplay between the autonomic nervous system and disease activity in multiple sclerosis (MS). We examined the relationship between heart rate variability (HRV), a reliable measure of vagal nerve function, and disease characteristics in a prospective MS cohort. Standard deviation of each normal-to-normal inter-beat interval (SDNN) and root mean square of successive differences (RMSSD), global indices of HRV, were measured in 114 MS patients, which included four predefined subgroups, and 30 age and sex-matched healthy controls (HC). We assessed group differences at baseline, HRV reproducibility at month 3, and used logistic regression modeling to relate baseline HRV with relapse occurrence. No significant HRV differences were found between MS and HC and between MS subgroups. In MS patients, both HRV indices correlated with age (r = −0.278, p = 0.018 and r = −0.319, p < 0.001, respectively) and with month 3 assessments (r = 0.695 and r = 0.760, p < 0.001). Higher SDNN and RMSSD at baseline were associated with self-reported relapses at month 3 (OR = 1.053, 95% CI (1.013–1.095), p = 0.009 and OR = 1.065, 95% CI (1.016–1.117), p = 0.009), and SDNN at baseline with relapses at month 12 (OR = 1.034, 95% CI (1.009–1.059), p = 0.008; ROC, AUC = 0.733, p = 0.002). There were no baseline HRV differences between MS and HC or between subgroups. Post-hoc analysis showed an association with an increased relapse risk.