Simple SummaryWe compared cortisol levels in the saliva of guide dogs and dogs that were trained as such but became companion dogs during a period of social isolation and exposure to a gunshot sound. The results showed that cortisol levels were higher in guide dogs than in companion dogs throughout the test. No changes were observed as a consequence of social isolation or exposure to the gunshot. This suggests that guide dogs maintain higher levels of basal cortisol compared with companion dogs, which could be associated with cognitive processes derived from working as guide dogs.AbstractGuide dogs work for extended periods and are exposed to multiple environmental stimuli that could lead to higher stress compared with companion dogs. Cortisol is the main hormone associated with stress in most mammals. This study included seven guide dogs and seven same-breed dogs that were trained as guide dogs but became companion dogs to compare their salivary cortisol levels before, during, and after a period of social isolation and exposure to a 110-decibel gunshot sound. Each dog was left alone in an empty room for 60 min. After 15 min, the dogs were exposed to the sound. We collected four saliva samples from each dog. The first one was taken 5 min before starting the social isolation period, and the following ones at 15, 30, and 45 min after the test started. A two-way ANOVA was used to compare the group effect and the time effect during isolation and noise exposure. The results showed higher levels of cortisol in the guide dogs compared with the companion dogs throughout the test. No differences were found in time or in the interaction between time and group. This suggests that being a guide dog increases levels of basal cortisol when compared with dogs that live as companion animals and family members.