Research on physiological stress and post-capture mortality of threatened species caught as bycatch is critical for the management of fisheries. The present study used laboratory simulations to examine the physiological stress response of sparsely spotted stingarees (Urolophus paucimaculatus) subjected to one of four different trawl treatments, including two different trawl durations as well as ancillary stressors of either air exposure or crowding. Physiological indicators (plasma lactate, urea, potassium and glucose) and changes in white blood cell counts were measured from blood samples taken throughout a 48 h recovery period. Mortality was low throughout this study (15% overall) and occurred only after >48 h following air exposure, crowding and 3 h trawl simulations. Plasma lactate, glucose and urea concentrations were identified as potential indicators of physiological stress, while plasma potassium and white blood cell counts were too variable to identify changes that would be expected to have biological consequences for stingarees. The characterization of the temporal profiles of physiological indicators facilitates a more accurate assessment of secondary stressors by identifying the best timing to sample stingaree blood when investigating post-capture stress physiology. High levels of lactate, increasing glucose and depressed urea were all recorded in response to air exposure following trawling, indicating that this is the primary source of stress in stingarees caught in trawling operations. These findings highlight the importance of improving bycatch sorting procedures to reduce the time out of the water for trawl-caught stingarees.