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Determining the number of familiarization sessions required for accurate recordings of ambulatory blood pressure monitoring and autonomic function is a prerequisite for the appropriate design of intervention studies. The benefit of familiarization trials remains largely unexplored. The objective of the current investigation was to assess the reproducibility of 24-h ambulatory blood pressure, 24-h heart rate variability (HRV) and resting measurements of HRV and blood pressure variability (BPV). Eleven prehypertensive and hypertensive adults participated. Ambulatory blood pressure and HRV were measured across 24 h on four occasions. In addition, 5-min resting measures of HRV and BPV were recorded and analysed. Variability between consecutive pairs of trials was calculated. The typical error induced by ambulatory recordings of systolic blood pressure reduced over time (3.8–2.8 mmHg). The greatest effect of familiarization was observed at night. Ambulatory HRV was more reproducible than resting measures. The most reproducible markers were root mean square of successive differences [coefficient of variation (CV): 13.2–10%] and high frequency normalized units (CV: 15.2–6.4%), with the percentage of adjacent NN intervals differing by more than 50 ms showing the poorest reproducibility (CV: 23.9–20.7%). Overall BPV (SD) was more reproducible than the frequency domain low frequency component. Familiarization trials are required for the most accurate recordings of both 24-h ambulatory blood pressure monitoring and HRV. Ambulatory HRV provide superior reproducibility to resting measurements.