Aim This study aimed to investigate the extent of autonomic nervous system dysfunction in patients with chronic migraine using heart rate variability analysis. In addition, we explored the potential association between heart rate variability and treatment outcomes in patients receiving preventive treatment. Methods In this cross-sectional and prospective study, we compared heart rate variability profiles in 81 preventive-naïve chronic migraine patients and 58 healthy controls. In addition, treatment responses of patients, who received a 12-week treatment with flunarizine, were assessed in relation to baseline heart rate variability. Results We observed that chronic migraine patients had a reduced heart rate variability, signifying autonomic dysfunction in comparison to healthy controls. Furthermore, patients presenting normal heart rate variability, characterized by a standard deviation exceeding 30 milliseconds in normal-to-normal RR intervals, experienced a superior response to flunarizine treatment. This improvement was exemplified by a significantly larger reduction in monthly headache days for patients with higher heart rate variability compared to those with lower heart rate variability: −9.7 (5.9) vs. −6.2 (6.0) days ( p = .026). Conclusions Autonomic dysfunction occurs in chronic migraine as evaluated by heart rate variability. A preserved function is associated with a better treatment outcome to flunarizine. Trial registration: Neurologic Signatures of Chronic Pain Disorders, NCT02747940. Registered 22 April 2016, https://clinicaltrials.gov/ct2/show/NCT02747940