The aim of this study was to assess the effect of body temperature (37 °C) on the cyclic fatigue resistance of three endodontic single-file systems using a new testing setup. One Shape® new generation (OS), WaveOne™ (WO) and WaveOne® GOLD (WOG), which are made from different NiTi alloys and operated in different motions (rotation/reciprocation), were evaluated. The study design included four groups. Each group comprised 30 files, 10 files of each of the three file systems, tested at 20 ± 2 °C (group 1 and 3) and at 37 ± 1 °C (group 2 and 4). All files were tested in a custom-made metal block with artificial canals of 60° angle, and a 5 mm and 3 mm radius of curvature, respectively. A heating element was attached to replicate a temperature of 37 °C. Files were introduced 18 mm into the canals and operated until failure. Transformation temperatures of five samples of each of the tested file systems were determined via the bend and free recovery (BFR) method. With the exception of WOG in canals with a 3 mm radius of curvature (p = 0.075), all the tested file systems showed statistically significantly less time needed to fracture when operated at 37 ± 1 °C compared to at 20 ± 2 °C in canals with a 5 mm and 3 mm radius of curvature using Mann-Whitney U test (p < 0.05). All file systems showed transformation temperatures below the body temperature. We concluded that body temperature directly affects the cyclic fatigue resistance of all tested file systems. Bend and free recovery can be suitable for the determination of austenite finish temperatures (Af) of endodontic instruments as it allows testing a longer portion of the instrument.