The analysis of heart rate variability (HRV) has become an established procedure in recent decades. Because there are no appropriate reference values available, HRV findings can still only be compared within a group or in individuals in longitudinal studies. The objectives of the present study were to examine a group of healthy subjects of different ages and sexes and to identify reference values for common HRV parameters. Long-term 24-hour electrocardiograms of 695 voluntary subjects were recorded by using a 2-channel Holter system over a period of 24 hours during daily activities. Reference values for men and women in 10-year age groups were calculated for standard deviation of NN intervals, root mean square of successive differences of NN intervals, standard deviation of the average of all consecutive 5-minute NN intervals, percentage of consecutive NN intervals that deviate from one another by more than 50 ms, low-frequency power normalized unit, high-frequency power normalized unit, low frequency/high frequency ratio, SD1, and SD2. The 5th and 95th percentiles were given for each sex and for the age groups 20-30, 30-40, 40-50, and 50-60 years. We observed a consistent decrease in HRV measures with increasing age as well as a sex dependency of HRV findings. We studied a large group of healthy subjects and identified reference values for commonly used HRV measures for 24-hour ECG measurements. The reference values differed considerably from the values published in 1996 in the Guidelines of the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. In the future, steps should be taken to expand the database and define reference values for the age groups under 20 and over 60 years. It would be desirable to obtain reference values for short-term recordings (eg, 5-minute recordings) as well.