Post-COVID-19 Syndrome (PCS) is a condition with multiple symptoms partly related to dysregulation of the autonomic nerve system. Assessment of heart rate variability (HRV) using 24 h Holter-ECG may serve as a surrogate to characterize cardiac autonomic activity. A prospective study including 103 PCS patients (time after infection = 252 days, age = 49.0 +/- 11.3 years, 45.7% women) was performed and patients underwent detailed clinical screening, cardiopulmonary exercise testing, and 24 h Holter monitoring. Data of PCS patients was compared to 103 CAD patients and a healthy control group (n = 90). After correction for age and sex, frequency-related variables differed in PCS patients compared to controls including LF/HFpower, LF/HFnu, and LF/HF ratio (24 h; p </= 0.001). By contrast, these variables were largely comparable between PCS and CAD patients, while sympathetic activation was highest in PCS patients during the 24 h period. Overall, PCS patients showed disturbed diurnal adjustment of HRV, with impaired parasympathetic activity at night. Patients hospitalized during acute infection showed an even more pronounced overactivation of sympathetic activity compared to patients who underwent ambulant care. Our data demonstrate persistent HRV alterations in PCS patients with long-term symptom duration, suggesting a sustained impairment of sympathovagal balance. Moreover, sympathetic overstimulation and diminished parasympathetic response in long-term PCS patients are comparable to findings in CAD patients. Whether HRV variables have a prognostic value in PCS and/or might serve as biomarkers indicating a successful interventional approach warrants further longitudinal studies.