Policing is a highly stressful and dangerous profession that involves a complex set of environmental, psychosocial, and health risks. The current study examined autonomic stress responses experienced by 64 police officers, during general duty calls for service (CFS) and interactions with the public. Advancing previous research, this study utilized GPS and detailed operational police records as objective evidence of specific activities throughout a CFS. These data were then used to map officers’ heart rate to both the phase of a call (e.g., dispatch, enroute) and incident factors (e.g., call priority, use-of-force). Furthermore, physical movement (i.e., location and inertia) was tracked and assisted in differentiating whether cardiovascular reactivity was due to physical or psychological stress. Officer characteristics, including years of service and training profiles, were examined to conduct a preliminary exploration of whether experience and relevant operational skills training impacted cardiovascular reactivity. Study results provide foundational evidence that CFS factors, specifically the phase of the call (i.e., arrival on scene, encountering a subject) and incident factors (i.e., call priority, weapons, arrest, use-of-force), influence physiological stress responses, which may be associated with short-term performance impairments and long-term health outcomes. Implications of research findings for operational policing, police training, and health research are discussed.