This study compared heart rate variability (HRV) parameters of cardiovascular autonomic regulation between well-trained masters and young cyclists at rest, during and following a continuous cycle (CTS) protocol. Ten masters (age = 56 ± 5years) and eight young (age = 26 ± 3years) cyclists completed a 100min experimental protocol consisting of a 60min CTS cycling bout at 95% of Ventilatory Threshold 2 followed by 40min of supine recovery. Beat-to-beat heart rate was measured continuously, and HRV parameters analysed at standardised 5min intervals during rest, exercise and recovery. The root mean square of the successive differences (RMSSD), low-frequency power and high-frequency power parameters were corrected by division of the R-R interval (time domain), or R-R interval squared (frequency domain). Further, the RMSSD and RMSSD:RR for successive 60-s R-R intervals at the onset (0-10min) and offset (60-70min) of CTS exercise were analysed over 10-min periods. The natural logarithm (Ln) of skewed parameters was taken for analysis. Significant interaction effects (P < 0.05) for 5min segments were found for (LnRMSSD, LnRMSSD:RR, LnHF and LnHF:RR2. Masters cyclists demonstrated lower values of parasympathetic activity at rest and during recovery (15-20min and 35-40min) compared to younger cyclists. However, similar HRV responses were observed during exercise, including throughout the acute onset and offset periods (P > 0.05). This data shows that cardiac autonomic regulation during, or immediately following CTS exercise may not be influenced by age, but masters athletes may exhibit a lower baseline for parasympathetic activity.