Context <p>Wildlife responses to disturbance may include physiological stress responses that are often difficult to observe yet important for wildlife health. Coupling physiological biologgers with tracking data can identify anthropogenic features associated with stress responses. Diagnosing when, where, and the response to stressors, along with how species may habituate or sensitize to stressors over time, is crucial for conservation efforts.</p> Objectives <p>We examined if mule deer (<i>Odocoileus hemionus</i>) exhibited acute stress responses to road crossings, identified the road features and contexts associated with the size of response, and determined factors that influence the response through repeated exposures over time.</p> Methods <p>We used GPS locations paired with cardiac monitors across three regions of Utah, USA to measure fine-scale changes in deer heart rates during road crossings. We measured dynamic traffic patterns using human mobility data (HMD) to characterize associated traffic volumes during crossing events.</p> Results <p>Road crossing events were associated with detectable heart rate spike (30.6&#xa0;bpm) and elevated mean heart rate (8.3%) in deer. Road crossings with higher HMD derived traffic volume resulted in greater heart rate spikes during migration (46%) and summer (24%) seasons. Habituation among deer correlated with higher frequency of crossings and better body condition, while sensitization correlated with higher traffic presence and higher road density.</p> Conclusions <p>Our findings highlight the stress response to roads and the factors driving the magnitude of the response. Integrating fine-scale physiological data with animal movement and human mobility data offers a means to diagnose dynamic stressors on the landscape at both broad and fine spatial and temporal scales for wildlife.</p>