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The purpose of this study was to assess the effects of sodium hypochlorite (NaOCl) immersion and sterilization on the cyclic fatigue resistance of heat-treated nickel-titanium (NiTi) rotary instruments.Two hundred ten new 25/.06 Twisted Files (TFs; SybronEndo, Orange, CA) and Hyflex CM (Coltene Whaledent, Cuyahoga Falls, OH) files were divided into 7 groups (n = 15) for each brand. Group 1 (control group) included new instruments that were not immersed in NaOCl or subjected to autoclave sterilization. Groups 2 and 3 were composed of instruments dynamically immersed for 3 minutes in 5% NaOCl solution 1 and 3 times, respectively. Groups 4 and 5 consisted of instruments only autoclaved 1 and 3 times, respectively. Groups 6 and 7 recruited instruments that received a cycle of both immersion in NaOCl and sterilization 1 and 3 times, respectively. Instruments were subsequently subjected to a fatigue test. The surface morphology of fractured instruments was studied by field-emission scanning electron microscopy and x-ray energy-dispersive spectrometric (EDS) analyses. The means and standard deviations of the number of cycles to failure (NCF) were calculated and statistically analyzed using 2-way analysis of variance (P < .05).Comparison among groups indicated no significant difference of NCF (P > .05) except for the groups of TFs sterilized 3 times without and with immersion in NaOCl (P < .05). HyFlex CM files exhibited higher cyclic fatigue resistance than TFs when files were sterilized 3 times, independently from immersion in NaOCl (P < .05). EDS analysis showed the presence of an oxide-rich layer on the Hyflex CM files' external surface. No morphologic or chemical differences were found between files of the same brand subjected to different treatments.Repeated cycles of sterilization did not influence the cyclic fatigue of NiTi files except for TFs, which showed a significant decrease of flexural resistance after 3 cycles of sterilization. Immersion in NaOCl did not reduce significantly the cyclic fatigue resistance of all heat-treated NiTi files tested.Hyflex CM cyclic fatigue was not affected by sodium hypochlorite (NaOCl) and/or sterilization.Three cycles of sterilization reduced the cyclic fatigue resistance of Twisted Files.The cyclic fatigue resistance of both instruments was not influenced by NaOCl.Hyflex CM had higher fatigue resistance than Twisted File after 3 sterilization cycles.Energy-dispersive spectrometric analysis showed the presence of an oxide-rich layer on the Hyflex CM files' surface.