Police work exposes officers to high levels of stress. Special emergency response team (SERT) service exposes personnel to additional demands. Specifically, the circadian cycles of SERT operators are subject to disruption, resulting in decreased capacity to compensate in response to changing demands. Adaptive regulation loss can be measured through heart rate variability (HRV) analysis. While HRV Trends with health and performance indicators, few studies have assessed the effect of overnight shift work on HRV in specialist police. Therefore, this study aimed to determine the effects overnight shift work on HRV in specialist police. HRV was analysed in 11 SERT officers and a significant (p = 0.037) difference was found in pRR50 levels across the training day (percentage of R-R intervals varying by >50 ms) between those who were off-duty and those who were on duty the night prior. HRV may be a valuable metric for quantifying load holistically and can be incorporated into health and fitness monitoring and personnel allocation decision making.