Ambassador animals are part of many zoo programs, but studies assessing their impact on these animals are relatively rare. This study validated an excrement glucocorticoid metabolite (GCM) assay for Magellanic penguins and used GCM measures in conjunction with behavioral observations to evaluate individual responses to participation in an ambassador animal program. Excrement samples and behavioral observations were collected daily from each bird during two phases, 1 week during which it participated in a twice-daily ambassador program and 1 week in which it did not. We found no differences in GCMs between phases or in comparisons between penguins with 5 or 10 years of program experience. GCM also did not show significant individual variation and did not increase over time during the program phase. There were no significant correlations between bird experience and behavior frequencies, nor GCM concentrations and behavior, across birds. We observed significant positive correlations between the penguins' engagement with novel objects during programs and their unguided approach to guests. Our results suggest that there is no adverse physiological effect of program participation on these penguins, that behavioral and physiological responses may be decoupled, and that choice and control can increase desired behaviors behavior during ambassador programs. K E Y W O R D S adrenocorticotrophic hormone (ACTH) challenge, corticosterone (CORT), fecal or excrement glucocorticoids, stress, welfare 1 | INTRODUCTION Connecting people with animals to prompt conservation-oriented behaviors is an integral component of many zoological missions. Animals presented regularly outside of their habitats to guests for education, outreach, or other special close-up programs are known as ambassador animals. Their use has been shown to increase audience engagement (Povey & Rios, 2002), positively impact visitor attitudes about conservation (Yerke & Burns, 1991), and influence future attitudes toward animals (Ballantyne et al., 2007;Kidd & Kidd, 1997;Kidd et al., 1995). Zoological organizations managing ambassador animal encounters should understand the effects these interactions have on individual animals and establish protocols that maximize both animal welfare and guest impact.Responses to stress are mediated by the hypothalamic-pituitaryadrenal (HPA) axis. During the cascade of reactions involved in the stress response, the hypothalamus secretes corticotrophin-releasing hormone, which stimulates the pituitary gland to secrete adrenocorticotrophic hormone (ACTH), which causes the adrenal cortex to release glucocorticoids (cortisol in most mammals and corticosterone in birds, reptiles, and rodents). When a stressor is detected, glucocorticoids secreted by the adrenal glands modulate physiological and behavioral responses to