Abstract The use of dogs in military work environments has always aroused great interest in the general population and determining the stress levels they go through is extremely important to maintain their welfare. The aim of this research was to evaluate if the work shifts in military working dogs leads to stress conditions and if this working influences on the reproductive performance and life quality. The study was conducted at the Military Police Kennel located at Campinas, Sao Paulo, Brazil. Eight male dogs of four different breeds (German Shepherd, Belgian Malinois Shepherd, Doberman, and Rottweiler) were evaluated during two different shifts: Working Shifts: animals working 12 hours a day with 2 hour-interval; and Control Shifts: animals that were on their day off (36 hours). Saliva samples were collected for cortisol analysis at the control and working shifts. The study was carried out over 60 days and analyzed behavior, physiology, and reproduction quality. Saliva samples, behavior observation of stereotyping, resting and moving activities and semen analysis were collected by digital stimulation (for combined second and third fractions). The salivary cortisol levels during the control and working shifts were between 0.361–0.438 and 0.312–0.592 µg/dL, respectively; the highest values were found at the end of working shifts. The animals were resting during most of the observation period, but few showed stereotypic behaviors. The testicular consistency was firm and semen parameters were within the normal values in German Shepherd, Belgian Malinois Shepherd, and Doberman dogs. However, Rottweiler dogs had a higher rate of sperm abnormalities, higher salivary cortisol levels, and more stereotypic behaviors. Nevertheless this work highlights the importance of further research relating reproduction and cortisol levels in military dogs.