Although ambulatory measurements of heart rate variability (HRV) are widely used, the reproducibility and seasonal variation of ambulatory sampled short-term HRV measurements in healthy participants has not been investigated before. In the present study we collected ambulatory ECGs from 19 healthy participants monthly for 12 months, and for a sub-group of 12 participants weekly for one month. Frequency-domain HRV-metrics were calculated for 5 min ECG segments during (i) a 15-min self-selected rest period (awake period), and (ii) a 30-min sleep period starting 45 min after estimated sleep onset. Total, within- and between-subject coefficient of variation (CV) and seasonal variation were estimated for ln (TP), ln (LFP), ln (HFP), ln (LF/HF), LFnu, HFnu, the mean heart period and the ECG derived respiratory frequency.The within- and between-subject CV varied considerably between different variables, from < 10% for ln (TP) and ln (LFP) to >100% for ln (LF/HF). Within- and between-subject CV of ln (HFP), LFnu and HFnu were 10-40%. A weak, but significant, seasonal variation was found for ln (TP) (p = 0.05), ln (LFP) (p<0.05) and the respiratory frequency (p<0.01), but the seasonal variation did not affect the within-subject CV. Furthermore, sample size calculations demonstrated that the reproducibility was sufficient for ambulatory HRV measurements to be used to study autonomic cardiac regulation in healthy populations.