Aim: The purpose of this study was to evaluate the effect of repeated autoclaving on the cyclic fatigue of NRT, Revo-S and ProTaper Universal rotary Ni-Ti instruments. Methods: The instruments selected for this study (n = 120) were divided into three groups according to the type of instrument: the NRT group, the Revo-S group and the ProTaper group. Each group (n = 40) was further subdivided into four subgroups according to the number of sterilisation cycles: subgroup A, in which the instruments were used without any exposure to sterilisation cycles; subgroup B (1 cycle); subgroup C (2 cycles); and subgroup D (3 cycles). All instruments were tested in simulated canals with a 60 degree angle of curvature and a 5 mm radius. The number of cycles to failure (NCF) was calculated and statistical analysis was carried out using the ANOVA test (P ≤ 0.05) and Bonferroni's post-hoc test. Scanning electron microscope analysis was used to analyse fractured instruments. Results: Statistically, significant differences were found between the NCF mean values of the three instruments when exposed to different sterilisation cycles (P 0.001). There was no statistically significant difference between the mean NCF amongst all subgroups in the ProTaper group. However, in the Revo-S and NRT systems, a statistically significant difference was found. The scanning electron microscope analysis of the separated instruments of all groups did not show any signs of plastic deformation on the flutes near the fracture site. Conclusion: The number of sterilisation cycles has a variable effect on cyclic fatigue depending on the type of Ni-Ti instrument. Palabras clave: autoclaving, cyclic fatigue, rotary nickel titanium instruments