This study investigated whether there is a relationship between heart rate variability (HRV) versus lifestyle and risk factors for cardiovascular disease in a population of healthy adolescents. HRV is as an index of tonic autonomic activity and in adults HRV is related to lifestyle and risk factors for cardiovascular disease, but it is not known if this is the case in adolescents. HRV was registered for 4 min in sitting position in 99 healthy adolescents (age range 15 years 11 months-17 years 7 months) and repeated after 6 months. On both occasions there were significant correlations (P < 0.05) between physical activity and HRV, with respective r values: high frequency (HF) 0.26, 0.30; low frequency power (LF) 0.35, 0.29 and the standard deviation of inter-beat intervals (SDNN) 0.28, 0.37. There was no significant interaction between first and second measurements. In contrast, there were no correlations to sleeping patterns, eating habits and smoking. Risk factors for cardiovascular disease [body mass index (BMI = weight (kg)/length in m(2)), systolic blood pressure and p-glucose] did not show any repeatable significant correlations to HRV. Multiple regression models showed that physical activity was a predictor for HF, LF and SDNN in both measurements. In conclusion HF, LF and SDNN were reproducible after 6 months and were related to physical activity on both occasions.