Repeated stressful events can negatively impact overall health by continuous stimulation of the hypothalamic–pituitary–adrenal axis, which leads to depletion of glucose stores and suppression of immune and reproductive function. The influence of stressors on survivability is particularly salient for coyote (Canis latrans) populations, because understanding how coyotes cope with stressors may provide relevant context on coyote adaptation to urbanized ecosystems. Our objectives were to physiologically validate fecal glucocorticoid metabolite (FGM) analysis in coyotes by performing an adrenocorticotropic hormone (ACTH) challenge in 12 captive individuals (6 treatment and 6 control) housed at the United States Department of Agriculture National Wildlife Research Center Predator Research Facility in Millville, Utah; to quantify potential changes in FGM output due to diurnal variation and sex; and to determine the effects of 2 anthropogenic events (placement of a novel cooling fan and state holiday celebrations) on the coyotes' stress responses (via FGM production). Results demonstrated that in response to the ACTH injection, treatment animals (3 males and 3 females) displayed FGM concentration peaks ≥ 5-fold (range: 5- to 30-fold) above their preinjection means approximately 8 h after injection, which was a greater (P = 0.037) response than control animals. FGM output was lowest for morning fecal samples compared with midday (P = 0.001) and evening (P < 0.001) samples. Within the evening period, FGM output for male samples tended to be higher (P = 0.056) than for female samples, although not significant. The anthropogenic events elicited FGM concentration peaks ≥ 5-fold above pre-event means for several of the study animals occurring approximately 12 and 9 h later, respectively. This study is the 1st to physiologically validate the measurement of stress physiology using FGM analysis in coyotes and demonstrate the impact of anthropogenic events on their stress response. Furthermore, this work provides a foundation for future studies of FGMs, stress, and anthropogenic effects in wild and captive systems.