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The shortnose sturgeon (Acipenser brevirostrum Lesueur, 1818) is a species of special concern in Canada, but little is known about their thermal biology. Information on the upper thermal tolerance of shortnose sturgeon becomes valuable for predicting future survival particularly with climate change and improving species management. Using a modified critical thermal maximum (CTmax) methodology, the objective is to determine whether previous thermal stress affects the thermal tolerance of juvenile shortnose sturgeon when exposed to a second thermal stress event. Prior exposure to thermal stress (CTmax1) did not affect the thermal tolerance (CTmax2) of juvenile shortnose sturgeon when a 24 h recovery period was allotted between tests. However, a significant increase in thermal tolerance occurred when the recovery time between the two thermal challenges was 1 h. Plasma glucose, lactate, and osmolality were all significantly affected by thermal stress, but values returned to control levels within 24 h. Hematocrit and plasma chloride concentrations were not significantly affected by thermal stress. All fish survived the CTmax testing. The data indicate that the thermal tolerance of juvenile shortnose sturgeon is modified when multiple thermal stresses occur closer together (1 h) but not if separated by a longer time period (24 h).