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Limnoperna fortunei (Dunker 1857) is a freshwater mussel with physiological tolerance to different environmental conditions, which may explain its success as an invasive species. The role of abiotic factors in its establishment, abundance and projections of risk of further spread into several areas has been studied. These mussels may respond to multiple environmental stressors, such as temperature, through physiological mechanisms, behavioral responses, mortality or some combination of these. The aim of this study was to investigate the behavioral responses (valve closing), glycogen concentrations and mortality of L. fortunei under four different temperatures (5°C, 10°C, 20°C and 30°C) during a chronic test (30 days). Two-way analysis of variance (ANOVA) was used to compare glycogen concentrations across days of the experiment and at the different temperatures. Differences in valve-closing behavior and mortality among temperatures were tested using repeated-measures ANOVA. We observed that most of the mussels maintained at 5°C closed their valves (74.7±15.3%), indicating that they remain inactive at low temperatures. The glycogen levels significantly differed among the temperatures tested. These differences occurred mainly due to the high glycogen values observed in mussels exposed to 10°C. Stability in glycogen concentrations was observed within each particular temperature. The cumulative mortality was higher at extreme temperatures (5°C and 30°C). The ideal temperature for laboratory maintenance and tests is approximately 20°C. Our data also show that L. fortunei can survive and maintain their energy reserves (glycogen) for several days at 5°C, an important feature related to its invasion success.