Introduction: The aim of this in vitro study was to determine the influence of two glide path preparation methods on the fracture rate of the Primary 25/08 WaveOne reciprocating instrument. The number of simulator Endo-Training-Blocks in each group were calculated at the time of instrument fracture and compared to determine the influence of glide path preparation on the failure rate of the Primary 25/08 WaveOne reciprocating instrument. Preparation times for different glide path methods and total time for root canal preparation, with or without glide path, was also calculated. Materials and Methods: Simulator Endo-Training-Blocks (n = 300) were selected and randomly divided into 3 groups (n = 100). The 3 groups were then sub-divided into 5 smaller groups (n = 20). Root canal preparation was done with new primary 25/08 WaveOne instruments allocated to each group (5 in total for each group, 1 for each sub-group) following different methods for glide path preparation. Group 1: no glide path (control); Group 2: glide path preparation with stainless-steel hand files (size 10 K-File followed by size 15 K-File and finally size 20 K-File until loose fitting); and Group 3: glide path preparation with rotary PathFiles (size 10 K-File until loose fitting followed by PathFile no.1 (ISO 13 tip), PathFile no.2 (ISO 16 tip) and finally PathFile no. 3 (ISO 19 tip)). The Primary 25/08 WaveOne reciprocating instrument was used with the WaveOne endodontic motor in “WaveOne All “mode with a brushing motion on the outstroke. Simulated root canals were irrigated with sterile water and Glyde was used as lubricating agent. The outcome was measured by recording how many simulators could be prepared with one instrument before instrument breakage occurred. The preparation protocol was repeated 5 times. The time it took to prepare the glide path and the total preparation time was also calculated. The data of the different parts of the project were collected and statistically analysed using the ANOVA / Bonferroni test. Results: Glide path preparation with PathFiles was significantly faster (13.3 s ± 2.60 s) than with hand files (25.1 s ± 1.70 s) (P < 0.001). The highest number of simulators could be prepared after glide path preparation was performed with PathFiles (19.2 ± 0.84) (P < 0.001). Pairwise comparisons at the Bonferroni adjusted significance level of 0.017 demonstrated that there were statistical significant differences (P < 0.001) when PathFiles (19.2 ± 0.84) and hand files (17.6 ±1.14) were compared to the no glide
path group (7.4 ± 0.89). There was no statistical significant difference between PathFiles and hand files groups. Total Preparation time was significantly shorter (P < 0.001) when an initial glide path was prepared with PathFiles (12.7 ± 0.22 s). The longest preparation time (P < 0.001) was calculated in group 1 where no glide path (21.2 ± 0.20 s) was present and the Endo-Training-Blocks were left undisturbed. Conclusion: Initial glide path preparation with PathFiles resulted in shorter preparation time (glide path and total preparation) and allowed a higher number of simulators prepared before failure of the WaveOne instrument.