Integrating thermal physiology with environmental temperature is essential to understanding distributions of species and vulnerability to climate change. Warming tolerance—the difference between an organism’s maximum thermal tolerance (T_max) and maximum habitat temperature (T_hab)—is frequently used to integrate organismal sensitivity and environmental exposure. Traditionally, applications of warming tolerance define T_max and T_hab as invariable magnitudes, yet tolerance magnitude depends on exposure duration and diel temperature cycles expose organisms to a range of temperature magnitudes and durations. How traditional (i.e., acute) estimates of warming tolerance compare to estimates from prolonged exposures remains poorly understood. In this study, magnitude-duration curves for tolerances of one cold-water, two cool-water, and one warm-water species of freshwater fish were compiled from the literature and compared to magnitude-duration exposures from 66 streams across the eastern United States. Warming tolerances were estimated for exposure durations spanning 0.01 to 24 hours. Current acute (0.01 hours) warming tolerances ranged from median 6.30°C for the cold-water species to 9.68°C for the warm-water species. The lowest warming tolerances corresponded to prolonged exposures lasting median 3.85 to 5.30 hours among species and were 2.51 to 4.38°C lower than acute estimates. Although acute estimates remained positive in historically occupied and unoccupied streams (6.30°C versus 2.33°C), estimates based on prolonged exposure were positive at occupied streams of the cold-water species but transitioned to negative in unoccupied streams (2.19°C versus -1.12°C). Acute warming tolerances for the cold-water species also remained positive under future climate (6.29 to 4.23°C) but approached zero at prolonged durations (2.19 to 0.09°C) and transitioned to negative for 47.2% of streams. Results demonstrate that acute measures of T_max and T_hab overestimate warming tolerances and therefore underestimate climate change vulnerability. Integrating magnitude-duration relationships into warming tolerance estimates can elucidate physiological mechanisms underlying species distributions and can improve accuracy of climate change vulnerability assessments.

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