Reduced short-term heart rate variability (HRV) is a risk factor for cardiovascular morbidity and total mortality. The reference values of short-term HRV indices in healthy young adults are unknown. To investigate age and sex differences in HRV and to generate reference values of short-term recordings, we examined 1780 healthy subjects aged 24 to 39 years. Both frequency and time domain HRV indices were computed. Indices included; low frequency (LF), high frequency (HF) and total components of spectral-HRV, the square root of mean squared differences of R-R-intervals and SD of normal R-R-intervals. Deep breathing test was performed and the mean ratio of R-R-intervals and the mean difference in instantaneous heart rate during breathing cycle were analyzed. Reproducibility of these indices was studied in 43 subjects. Aging and higher heart rate were inversely associated with all HRV indices (all p values <0.0001). Women had higher HF and lower LF compared to men (both p<0.0001). Women had higher resting heart rate (70 vs. 65 bpm, p<0.0001). The reproducibility of HRV indices and deep breathing test were good (CV 5.3-13.9%). We conclude that age, sex and heart rate needs to be considered when evaluating HRV indices and when generating reference values. Because of good reproducibility the short-term indices of HRV and deep breathing test can be used in clinical work.