Aim: The aim of this systematic review was to compare the effect of body temperature (I) on the cyclic fatigue resistance (O) of nickel–titanium (NiTi) endodontic instruments (P) to that of room temperature (C). Methods: The study was registered in the PROSPERO database (CRD42020204286). A systematic search in PubMed, Scopus, Web of Science, Google Scholar, and OpenGrey was conducted in English until December 31, 2021. In vitro studies comparing the cyclic fatigue resistance of NiTi instruments at the body (35°C ± 2°C) and room temperature (20°C–25°C) were included. Eligible studies were evaluated for risk of bias and meta-analyzed to estimate the effects. Results: Twenty-one studies out of 347 met the criteria for inclusion. The meta-analysis included six studies ( n = 215) with comparative study parameters. The overall effect sizes (5.49; 95% confidence interval [CI]: 4.04–6.93) were significantly different ( P < 0.001), indicating that the mean values at room temperature were significantly ( P < 0.001) higher. The effect sizes for full rotary motion (standardized mean difference [SMD]: 4.80; 95% CI: 3.04–6.56) and reciprocating motion (SMD: 6.37; 95% CI: 3.63–9.11) were not significantly different ( P = 0.346). Heterogeneity was high ( I 2 = 94%). Sensitivity analysis revealed that the SMD values were not significantly different ( P > 0.05) from the overall effect size, indicating that none of the studies had an effect on the overall effect size. Conclusions: Within the limitation of the study, the cyclic fatigue resistance of heat-treated NiTi endodontic files is significantly reduced at body temperature when compared to room temperature. Cyclic fatigue testing should be conducted at simulated body temperature.