Objective The aim of this study was to compare the cyclic fatigue resistance at body temperature and phase transformation behaviors of novel Rotate instrument (25.06) with rotating Mtwo (25.06) and reciprocating Reciproc Blue (25.08) and Reciproc (25.08) instruments. Materials and methods The Rotate, Reciproc Blue, Reciproc, and Mtwo instruments free of visible deformations were collected and tested in a static cyclic fatigue test method, which has a ceramic block containing an artificial canal with 60 degrees angle of curvature and a 5-mm radius of curvature at 37 degrees C (n= 16). All instruments were operated until fracture occurred, and both time to fracture (TF) and the lengths of the fractured fragments were recorded. TF data was analyzed with one-way ANOVA followed by post hoc Tukey tests and Weibull analysis, and fractured fragment length data were subjected to one-way ANOVA and post hoc Tukey tests (P< 0.05). Two unused instruments from each brand were also subjected to differential scanning calorimetry (DSC) analysis to determine their phase transformation temperatures. Results The Reciproc Blue instruments showed significantly higher TF values and reliability than the other groups (P< 0.05). Rotate instruments exhibited greater cyclic fatigue resistance than the Reciproc and Mtwo instruments (P< 0.05). No significant difference was detected among the fractured fragment lengths (P> 0.05). The lowest austenite finish temperature was exhibited by the Mtwo, which presented a single transformation peak, followed by the Rotate, Reciproc Blue, and Reciproc instruments which all presented two peaks during transformation. Conclusions Cyclic fatigue resistance of instruments manufactured from thermally treated Blue wire instruments was superior to those of the Mtwo and Reciproc, whereas reciprocating the Blue wire showed the highest resistance.