The purpose of this study was to evaluate the effect of simulated clinical use on the cyclic fatigue of TF and to examine fracture characteristics of fractured segments using SEM.A total of 54 TF (18 sets) were used to prepare moderately curved root canals. Each set had three file sizes (25/ 0.08 - 25/ 0.06 - 30/ 0.06). The files were divided into control group or unused group and experimental group. The experimental group was divided into three subgroups according to the number of canals that was prepared, 3, 6 and 9 canals. After clinical use, the TFs were rotated in stainless steel canals until fracture. The artificial canals for cyclic fatigue testing were made with a tapered shape corresponding to the dimensions of the tested TF with an angle of curvature of 60 degrees and radius of curvature of 5 mm. the files then tested in cyclic fatigue testing device to determine their cyclic fatigue.The results showed statistically significant difference (P<0.05) in cycles to failure between new and used (control group and experimental group) as revealed by two way ANOVA test. Pair-wise Newman-Keuls post-hoc tests revealed that the difference in NCF between three canals subgroup and six canals subgroup was statistically non-significant (p>0.05). The difference between control group and each subgroup and between other subgroups were statistically significant (p<0.05). Regardless the number of canals prepared, two way ANOVA test showed statistically significant difference in NCF mean values between TF sizes (P<0.05). Pair-wise Newman-Keuls post-hoc tests revealed that the difference between 25/0.08 TF and 30/0.06 TF was statistically non-significant (p>0.05) while the difference between 25/0.06 TF and the other TF sizes (25/0.08 TF and 30/0.06 TF) was statistically significant (p < 0.05). Scanning electron microscopy evaluation of test samples revealed that the fracture surfaces showed the characteristic features of ductile fracture.Within limitations of this study, we found that simulated clinical use significantly reduced the cyclic fatigue resistance of TF size 25/0.06 compared to unused files of the same instrument size when used in moderately curved canal while the other two files (25/0.08 and 30/0.06) showed more resistance to cyclic fatigue. Each TF instrument was successfully operated without any file sparation, demonstrating that TF rotary instruments can be used safely in clinical conditions up to 9 times in moderately curved molar teeth.