Background This nested case-control study can be viewed as an efficient way to sample subjects from a large cohort study case-control study aimed to analyze the effect of different clinical factors on the appearance of vertical root fractures in endodontically-treated teeth (ETT) over time. Material and Methods By matching 90 cases and 270 controls nested in a cohort of 450 patients. Incident “cases” included those ETT in which a confirmed VRF. The “controls” were ETT with clinical and radiographic evidence of normality. When an “incident case” was detected, three random “controls” according to the evaluation time registered in years were selected. Time interval corresponded to the exposure time from the end of the endodontic treatment until the tooth was included in the study. Demographic and clinical parameters included: age, gender, type, and location of the tooth, type of endodontic treatment, number of appointments necessary to complete the endodontic treatment, use of intra-canal medication, the apical extension of the filling, type of coronal restoration, the role of the tooth in the rehabilitation treatment, presence of intra-radicular posts, and presence of an adjacent implant, were analyzed over time. Statistical analysis: univariate descriptive analysis, Pearson’s χ2 test, and a logistic regression model adjusted for the most significant variables with a 95% confidence interval. Results The prevalence of vertical root fractures was 16.42%. The multivariate analysis confirmed that re-treatment (OR:12.19; OR:4.28; P <0.05) lasting five to ten years and intra-canal medication (OR:6.16; P =0.004) for more than eleven years significantly more associated with the risk of vertical root fracture. For teeth with intra-canal post or direct coronal restorations, the risk of vertical root fracture was three times lower. Conclusions Endodontic re-treatment and the use of intracanal medication such as calcium hydroxide should be considered primary and secondary risk factors, respectively, according to the appearance of VRF over time. Key words: Apical surgery, endodontic re-treatment, endodontically-treated teeth, risk factors, vertical root fracture.