Background: Leopard sharks (Triakis semifasciata) are abundant, coastal, eastern Pacific, mesopredatory sharks and are frequently managed in aquariums and zoos. Medical examinations are a routine part of good husbandry practices, but the handling protocols vary greatly between facilities. In this study, we compared the physiological stress responses of T. semifasciata associated with manual restraint and sedated handling under parallel holding and handling procedures in a 10 min interval. Methods: Blood was collected and analyzed for the hematocrit (Hct), lactate, glucose, beta-hydroxybutyrate (β-HB), pH, blood gasses, 1α-hydroxycorticosterone (1α-OHB), and corticosterone (B). Results: Overall, a minimal physiological stress response was observed in both groups. However, statistically significant increases in the PCO2, β-HB and lactate occurred, while the pHTC, SO2, and BE decreased over time in the manual-restraint group. In the sedated group, the B, PCO2, and lactate increased while the pHTC, SO2, and base excess (BE) decreased over time. When comparing treatments, the sedated group had a higher B and PCO2 TC and a lower pHTC, BE, and SO2 than the manual-restraint group at the second time point. Conclusions: This study indicates that manual restraint is comparable to the described sedation in T. semifasciata for minimally invasive procedures. The overall results show that this species had low physiological stress responses to both methods.