In vitro studies investigating the mechanical properties of dental reconstructions use various materials to replicate prepared teeth. However, no uniform recommendation exists as to which material is most suitable for standardized testing. The purpose of this study was to identify a material that resembles human dentin in fracture load tests. Sixteen human teeth were scanned with an intraoral scanner to obtain copies of the original crown morphology and were then prepared for crowns. Replica dies of the prepared teeth including the root morphology were fabricated with a Computer-aided design and computer-aided manufacturing (CAD/CAM) system and divided into four groups: (A) reinforced composite (RC); (B) human dentin (HD); (C) polymethyl methacrylate (PM); and (D) hybrid ceramic (HC). Sixty-four feldspar ceramic crowns were designed with the biocopy mode, fabricated with a CAD/CAM system, luted on the dies, and then with the roots embedded in polymethyl methacrylate. Care was taken to position all specimens of the same morphology identically. Thermo-mechanical load cycling was performed in a chewing simulator followed by fractural loading of the crowns. A mixed effect linear model was fitted to the data, and pairwise contrasts were estimated on the marginal means and corrected for multiple testing according to Tukey (α = 0.05). The means for fracture load (N) were 2435 N (95% CI (2162, 2709)) for hybrid ceramic, 1838 N (95% CI (1565, 2112)) for reinforced composite, 1670 N (95% CI (1396, 1943)) for human tooth and 1142 N (95% CI (868, 1415)) for polymethyl methacrylate abutment materials. Post-hoc pairwise contrasts revealed a statistically significant (p < 0.05) difference among all groups except for reinforced composite and human dentin (p = 0.76). The results indicate that the mechanical properties of abutment dies play a significant role for a possible substitution of natural teeth in in vitro studies.