Climate change has resulted in increased incidence and variability of warming episodes in cold-water streams that support salmonids. The capacity to acclimate to warm temperatures may allow cold-water fish to persist in spite of changing thermal regimes, but accurately predicting fish performance under fluctuating stream temperatures also requires understanding re-acclimation to cool water, which is less well understood. We tested how thermal acclimation to warm temperatures and re-acclimation to cool water affected thermal tolerance and physiological endpoints in juvenile brook trout (Salvelinus fontinalis). We show that an initial thermal exposure (22°C, ΔT = 7°C) of 3, 7 and 14 days (but not 1 day) improved critical thermal maximum (CT_max) after a 14-day re-acclimation to cooler temperatures (15°C). Fish growth during the re-acclimation period decreased with increasing duration of initial thermal exposure (22°C). Physiological parameters associated with thermal acclimation (cortisol, glucose, haematocrit and haemoglobin) were lower at 15°C re-acclimation temperature than at the initial thermal treatment (22°C) and in some cases, lower than the 15°C control. Muscle HSP70 protein increased early (1 day) as part of the warm acclimation process and remained elevated at lower levels for up to 14 days. During re-acclimation to 15°C, HSP70 decreased relative to initial measures at 22°C. Fish exposed to the longest thermal treatment (22°C for 14 days) maintained elevated CT_max after 30 days of re-acclimation to 15°C without observed differences in the measured physiological endpoints but returned to control levels after 42 days at 15°C. This work shows that high-temperature acclimation effects in brook trout are retained for up to 30 days following re-acclimation to cool temperatures, and that isolated warming events may be expected to temporarily enhance thermal tolerance in subsequent thermal challenges.