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Abstract The mysid Neomysis americana (Smith, 1873) is native to shallow shelf waters and estuaries of the western Atlantic coast of North America. Despite the important role mysids such as N. americana play in estuarine ecosystems as both consumers and as prey for higher trophic levels, there is limited information on how metabolism influences their spatial ecology and habitat requirements. In tributaries of Chesapeake Bay, MD, USA, previous research has shown that summer water temperatures can approach the lethal upper tolerance limit for N. americana. We measured the per capita metabolic rate (µgO2 min–1) of N. americana from the upper Patuxent River near Benedict, MD, a tributary of Chesapeake Bay in the laboratory to evaluate the metabolic response to salinity and temperature conditions that mysids experience in natural habitats. Sex-specific and diel patterns in metabolic rate were quantified. Metabolic rates did not differ between night and day and there was no significant difference in metabolic rate between males and females, exclusive of gravid females. Metabolic rates were lowest in salinity treatments of 2 and 8 at 29 °C, and highest in the salinity 2 treatment at 22 °C. Only temperature had a statistically significant, albeit unexpected, effect. This study shows that the metabolic response of N. americana to temperature and salinity conditions is complex and plastic, and that metabolic rates can vary 3–4 fold within realistic summer temperature and salinity conditions. As environmental conditions continue to change, understanding metabolic response of mysids to realistic salinity and temperature conditions is necessary for understanding their distributions in temperate estuaries.