OBJECTIVE: To establish reference values of heart rate variability (HRV) measures in a cohort of adolescent boys and to determine the relationship between HRV and the clustering of risk factors (RFs) for cardiovascular disease. STUDY DESIGN: This cross-sectional study included 1152 adolescent boys (age: 16.6 +/- 1.2 years old). Demographic data, health-related habits, obesity indicators, and blood pressure were evaluated. HRV measures of time (SD of all RR intervals, root mean square of the squared differences between adjacent normal RR intervals, and the percentage of adjacent intervals over 50 ms) and frequency domains were assessed (low [LF] and high [HF] frequency). RESULTS: The components of HRV were RR interval (827 +/- 128 ms), SD of all RR intervals (61.9 +/- 23.5 ms), root mean square of the squared differences between adjacent normal RR intervals (54.5 +/- 29.4 ms), percentage of adjacent intervals over 50 ms (29.4 +/- 20.4%), LF (53 +/- 16 nu), HF (47 +/- 16), and LF/HF (1.44 +/- 1.08). Greater sympathetic and lower parasympathetic modulation at rest were associated with higher adiposity, higher blood pressure and physical inactivity. Adolescents with 2 or more RFs also presented lower HRV than subjects with no RFs (P < .001). CONCLUSIONS: The study has provided descriptive indicators that help the interpretation of HRV results in adolescents. Lower HRV measures are associated with the clustering of cardiovascular RFs.