We aimed to determine the association of a comprehensive healthy lifestyle with heart rate variability (HRV), a validated measure of autonomic function. This was a prospective cohort study. A population-based sample of 2079 individuals aged 25-41 years without prevalent cardiovascular disease was investigated. The standard deviation of all normal RR intervals (SDNN) during 24-hour electrocardiography was used as main HRV marker. Healthy lifestyle metrics were summed to a validated lifestyle-score ranging from 0 = most unhealthy to 7 = most healthy. One point was given for each of the following items: never smoking cigarettes; consuming a healthy diet; performing moderate (≥150 min/week) or vigorous (≥75 min/week) physical activity; body mass index (BMI)<25 kg/m(2); total cholesterol<200 mg/dl; glycated haemoglobin A1c<5.7%; and blood pressure<120 (systolic) and <80 mm Hg (diastolic). Median age of the participants (47% males) was 37 years. Mean SDNN was 153 ms and median lifestyle-score was four. A score of 0/1 or 6/7 was found in 5.2% and 11.0%, respectively. In multivariable linear regression analysis with SDNN as the outcome variable, the β-estimate (95% confidence interval (CI)) for a one-point increase of the lifestyle-score was 0.14 (0.11-0.17), p < 0.0001. This relationship was attenuated but remained significant after additional adjustment for resting heart rate (HR) (β-estimate (95% CI) 0.07 (0.07-0.10), p < 0.0001) or 24-hour HR (0.04 (0.01-0.07), p = 0.003). Few individuals adopted a healthy lifestyle in this large contemporary cohort of young adults from the general population. Adopting a healthy lifestyle has an important effect on autonomic function.