Background. This study was conducted to compare the cyclic fatigue resistance of VDW.ROTATE,TruNatomy Prime, HyFlex CM, and 2Shape nickel-titanium (NiTi) rotary instruments in doublecurvedcanals in a simulated clinical environment. Methods. Eighty NiTi files were used for the fatigue testing in stainless steel canals compatible withinstrument sizes until fracture occurred (n=20): VDW.ROTATE (04./25#), TruNatomy Prime (04./26#),HyFlex CM (04./25#) and 2Shape TS04./25#( 1). For each instrument, the number of cycles to fracture(NCF) was calculated, and the fractured fragment length (FL) was measured. To verify that the fileswere fractured due to cyclic fatigue, the fractured surfaces of the files were evaluated under a scanningelectron microscope. Data were statistically analyzed using the Kruskal–Wallis and Student’s t-tests atthe %95 confidence level. Results. The failure of the files due to cyclic fatigue was first seen in the apical curvature before thecoronal curvature (P<0.05). The highest fatigue resistance was observed in VDW.ROTATE and HyFlexCM files in both curvatures (P<0.05). There were no significant differences in the fatigue resistancebetween the HyFlex CM and VDW.ROTATE files or between the 2Shape and the TruNatomy files(P>0.05). There was no difference in the fractured lengths of the files between the apical and coronalcurvatures (P>0.05). Conclusion. In artificial S-shaped root canals, VDW.ROTATE and HyFlex CM files exhibited higherfatigue resistance compared to 2Shape and TruNatomy files.