Northern (Glaucomys sabrinus) and southern (G. volans) flying squirrels have glucocorticoid (GC; stress hormone) levels higher than most vertebrates but virtually no binding capacity for these GCs via the carrier protein, corticosteroid-binding globulin. Thus, their total GCs are essentially all free and biologically active. However, the GC estimates come from blood samples taken after squirrels had been in live traps, and thus in a stress-induced state. Obtaining baseline values for physiological variables is valuable for assessing the response of vertebrates to stressors in their environment. We compared baseline plasma total cortisol levels (within 3 min of capture) to stress-induced levels (after 30 min of trap restraint) in both flying squirrel species. We recorded baseline cortisol levels that were some of the highest ever reported for mammals, indicating their stress axes operate at a higher set point than most other species. As part of the stress response, we also measured 4 indices in addition to cortisol. Total cortisol and free fatty acids increased in both species, as predicted. In contrast with our predictions, blood glucose and neutrophil/lymphocyte ratio showed no overall change, and hematocrit decreased significantly. New World flying squirrels therefore appear to have a stress response that differs from many other mammals. The selective forces driving the physiology of these animals remain elusive, but this lineage may provide an interesting comparative system for the study of stress axis function and its evolution among vertebrates.