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This study examined the effect of different temperatures on the cyclic fatigue of nickel-titanium rotary files.Three groups of nickel-titanium rotary files (EF group [EdgeFile; EdgeEndo, Albuquerque, NM], VB group [Vortex Blue; Dentsply Tulsa Dental Specialties, Tulsa, OK], and ESX group [ESX; Brasseler USA, Savannah, GA]) of size 25 with a .04 taper and 25-mm length were tested in a metal block that simulated a canal curvature of 60° and a 5-mm radius curvature. The block was submerged in a water bath filled with water at 3°C, 22°C, 37°C, and 60°C. At each temperature, 30 files from each group were rotated at 500 rpm in the block. The number of cycles to fracture (NCF) was calculated. Statistical analysis was completed using a 1-way analysis of variance with significance at P < .05.VB group showed a significant decrease in NCF as the temperature increased from 3°C to 60°C. The ESX group showed a significant decrease in NCF as the temperature increased from 3°C to 37°C. The EF group showed a significant increase in NCF from 3°C to 22°C and a significant decrease in NCF from 22°C to 37°C. For each temperature, the EF group showed higher NCF than the VB group, which showed higher NCF than the ESX group.In this in vitro study, temperature was found to significantly affect the cyclic fatigue of nickel-titanium rotary files. At each tested temperature, NCF was the highest for the EF group followed by the VB group and lowest for the ESX group. Future cyclic fatigue studies should be conducted at body temperature.We tested the number of cycles to fracture of 3 different nickel-titanium rotary files at 4 different temperatures.Overall, the number of cycles to fracture was significantly less at warmer temperatures than at cooler temperatures.Testing at body temperature caused a significant decrease in the number of cycles to fracture compared with testing at room temperature.This is important because most cyclic fatigue studies are conducted at room temperature, so it is suggested for future studies to be conducted at body temperature.