Objective: The aim of this systematic multiscale analysis was to evaluate the effects of thermoforming on the physical and mechanical properties of thermoplastic materials used to fabricate transparent orthodontic aligners (TOAs). Methods: Specimens were fabricated using four types of thermoplastic materials with different thicknesses under a thermal vacuum. Transparency, water absorption and solubility, surface hardness, and the results of three-point bending and tensile tests were evaluated before and after thermoforming. Data were analyzed using one-way analysis of variance and Student’s t -test. Results: After thermoforming, the transparency of Duran and Essix A+ decreased, while the water absorption ability of all materials; the water solubility of Duran, Essix A+, and Essix ACE; and the surface hardness of Duran and Essix A+ increased. The flexure modulus for the 0.5-mm-thick Duran, Essix A+, and eCligner specimens increased, whereas that for the 0.75-/1.0-mm-thick Duran and eClginer specimens decreased. In addition, the elastic modulus increased for the 0.5-mm-thick Essix A+ specimens and decreased for the 0.75-mm-thick Duran and Essix ACE and the 1.0-mm-thick Essix ACE specimens. Conclusions: Our findings suggest that the physical and mechanical properties of thermoplastic materials used for the fabrication of TOAs should be evaluated after thermoforming in order to characterize their properties for clinical application.