Objective: To measure the fracture resistance of mandibular premolar roots following root canal instrumentation with different sizes. Methods: A total of 100 human permanent mandibular premolars with a straight single canal were decoronated and assigned to 10 groups (n = 10) by block randomisation. In the control group, the roots were uninstrumented, whereas roots in the nine experimental groups were instrumented to different master apical files (MAF) and tapers (MAF/taper): 40/0.05, 45/0.05, 50/0.05, 55/0.05, 60/0.05, 40/0.10, 40/0.15, 45/0.10 and 45/0.15. All roots were subjected to vertical loading until fracture. Results: Fracture load values for instrumented roots were lower than the intact roots of the control group. In 50/0.05, 55/0.05, 60/0.05, 40/0.15 and 45/0.15 groups, the fracture load values were significantly lower than the fracture load value for the control group (P 0.05) with a 30% decrease. No significant differences in the fracture modes were detected among the 10 groups (P > 0.05). Conclusion: Mechanical instrumentation adversely affects the fracture resistance of roots. When the roots of mandibular premolars were instrumented to a MAF equal to or larger than 50 with a taper of 0.05 or to a MAF of 40 or 45 with a taper of 0.15, the fracture load values significantly decreased. Keywords: tooth fractures, root canal preparation, mandibular premolars