Recent research has elucidated the acute lethal effects of elevated water temperatures to glochidia (larvae), juvenile, and adult life stages of freshwater mussels (Order Unionida), but few studies have focused on sublethal effects of thermal stress. We evaluated the sublethal effects of elevated temperature on burrowing behavior and byssus production in juveniles, and on enzymatic biomarkers of stress in adults in acute (96 h) laboratory experiments in sediment, with two acclimation temperatures (22 and 27 °C) and two experimental water levels (watered and dewatered) as proxies for flow regime. Increasing temperature significantly reduced burrowing in all five species tested, and the dewatered treatment (a proxy for drought conditions) reduced burrowing in all but Amblema plicata. Production of byssal threads was affected most drastically by flow regime, with the probability of byssus presence reduced by 93–99% in the dewatered treatment, compared to the watered treatment (a proxy for low flow conditions); increasing temperature alone reduced byssus by 18–35%. Alanine aminotransferase and aspartate aminotransferase were significantly affected by treatment temperature in the 27 °C acclimation, watered test (p = 0.04 and 0.02, respectively). Our results are important in the context of climate change, because stream temperature and flow are expected to change with increasing air temperature and altered precipitation patterns.