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ABSTRACT Torsional overload is a fracture representative parameter for instruments in single-file techniques. Objective The aim of this study was to assess the influence of clinical use, in vivo, on the torsional behavior of Reciproc and WaveOne instruments considering the possibility that they degraded with use. Material and Methods Diameter at each millimeter, pitch length, and area at 3 mm from the tip were determined for both types of instruments. Twenty-four instruments, size 25, 0.08 taper, of each system were divided into two groups (n=12 each): Control Group (CG), in which new Reciproc (RC) and WaveOne Primary (WO) instruments were tested in torsion until rupture based on ISO 3630-1; and Experimental Group (EG), in which each new instrument was clinically used to clean and shape the root canals of one molar. After clinical use, the instruments were analyzed using optical and scanning electron microscopy and subsequently tested in torsion until fracture. Data were analyzed using one-way analysis of variance at a=.05. Results WO instruments showed significantly higher mean values of cross-sectional area A3 (P=0.000) and smaller pitch lengths than RC instruments with no statistically significant differences in the diameter at D3 (P=0.521). No significant differences in torsional resistance between the RC and WO new instruments (P=0.134) were found. The clinical use resulted in a tendency of reduction in the maximum torque of the analyzed instruments but no statistically significant difference was observed between them (P=0.327). During the preparation of the root canals, two fractured RC instruments and longitudinal and transversal cracks in RC and WO instruments were observed through SEM analysis. Conclusion After clinical use, no statistically significant reduction in the torsional resistance was observed.