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This study aimed to analyze the effects of ultrasonic removal of fractured files from the middle third of root canals on the vertical root fracture resistance.This study was an extension of a previous study assessing the effects of fractured file removal on dentinal microcracks. It included 18 bilaterally matched pairs of human mandibular incisors prepared and analyzed in the previous study. Briefly, 1 member of each pair was assigned to an ultrasonic or a control group. In the ultrasonic group, K-files were fractured in the middle third of canals followed by ultrasonic file removal and canal preparation. In the control group, the root canals were simply prepared. Micro–computed tomographic imaging was performed before and after treatment, and the cross-sectional root images were screened for microcracks. For the present study, 3-dimensional reconstruction was performed for volumetric assessments. The fracture resistance was measured using a universal testing machine. All data were statistically analyzed.Fracture loads were significantly smaller in the ultrasonic group (P < .05). The percentage increase in the canal volume significantly influenced the root fracture resistance (P < .05), whereas microcracks had no significant effect (P > .05).Ultrasonic removal of fractured instruments from the middle third of root canals lowers the vertical root fracture resistance, with increased dentin removal being the primary cause.