OBJECTIVE This study sought to examine clinical determinants of heart rate variability and to report normative reference values for eight heart rate variability measures. BACKGROUND Although the clinical implications of heart rate variability have been described, clinical determinants and normative values of heart rate variability measures have not been studied systematically in a large community-based population. METHODS The first 2 h of ambulatory electrocardiographic recordings obtained in Framingham Heart Study subjects attending a routine examination were reprocessed for heart rate variability. Recordings with transient or persistent nonsinus rhythm, premature beats > 10% of total beats, < 1-h recording time or processed time < 50% of recorded time were excluded; subjects receiving antiarrhythmic medications also were excluded. Among five frequency domain and three time domain measures that were obtained, low frequency power (0.04 to 0.15 Hz), high frequency power (0.15 to 0.40 Hz) and the standard deviation of total normal RR intervals based on 2-h recordings were selected for the principal analyses. Variables with potential physiologic effects or possible technical influences on heart rate variability measures were chosen for multiple linear regression analysis. Normative values, derived from a subset of healthy subjects, were adjusted for age and heart rate. RESULTS There were 2,722 eligible subjects with a mean age (+/-SD) of 55 +/- 14 years. Three separate multiple linear regression analyses revealed that higher heart rate, older age, beta-adrenergic blocking agent use, history of myocardial infarction or congestive heart failure, diuretic use, diastolic blood pressure > or = 90 mm Hg, diabetes mellitus, consumption of three or more cups of coffee per day and smoking were associated with lower values of one or more heart rate variability measures, whereas longer processed time, start time in the morning, frequent supraventricular and ventricular premature beats, female gender and systolic blood pressure > or = 160 mm Hg were associated with higher values. Age and heart rate were the major determinants of all three selected heart rate variability measures (partial R2 values 0.125 to 0.389). Normative reference values for all eight heart rate variability measures are presented. CONCLUSIONS Age and heart rate must be taken into account when assessing heart rate variability.