Aim To evaluate the effect of multiple refreshment/activation cycles and temperature on the reaction rate of sodium hypochlorite (NaOCl) with bovine dentine during ultrasonic activated irrigation (UAI) under laboratory conditions. Methodology The root canal walls of 24 standardized root canals in bovine incisors were exposed to a standardized volume of NaOCl at different temperatures (24 °C and 38 °C) and exposure times (20, 60 and 180 s). The irrigant was refreshed and ultrasonically activated four times for 20 s followed by a 40 s rest interval, with no refreshment and no activation as the controls. The reaction rate was determined by measuring the amount of active chlorine in the NaOCl solution before and after being exposed to dentine during the specific experimental conditions. Calorimetry was used to measure the electrical-to-sonochemical conversion efficiency during ultrasonic activation. Results Refreshment, activation and exposure time all increased the reaction rate of NaOCl (P < 0.05). During activation, the temperature of the irrigant increased up to 10 °C. Such temperature rise was insufficient to enhance the reaction rate of NaOCl (P > 0.125). Conclusions The reaction rate of NaOCl with dentine is enhanced by refreshment, ultrasonic activation and exposure time. Temperature rise of irrigant during ultrasonic activation was not sufficient to alter the reaction rate.