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Background: To compare and investigate the effects of intraoral ageing on the thickness of one group of directly printed and two groups of thermoformed aligners on the labial surface of maxillary central incisors., Materials and Methods: Six groups (12 samples per group) were included in this prospective in vivo experiment. Groups DP-Clin, INV-Clin and CA-Clin consisted of directly printed (Tera Harz TC-85 DAC resin), thermoformed (Invisalign, PU based polymer) and in house thermoformed (CA-Pro, PET-G based polymer) aligners, retrieved after 1 week of intraoral service. Groups DP-Ctr, INV-Ctr and CA-Ctr included unused aligners samples. Thickness measurements were conducted using confocal laser scanning microscopy (CLSM). Data that underwent log-10 transformation was analysed by multiple linear regression analysis (p < .05)., Results: Statistically significant differences were found between the materials in both Clin and Ctr categories (p < .001). Group DP had the highest thickness among the groups and the least thickness was observed in the CA group (p < .001). However, intraoral ageing did not significantly affect the aligner thickness of any groups., Conclusions: Both thermoforming and direct printing of clear aligners led to thickness deviations in terms of increase for printed aligners and decrease for thermoformed aligners. Intraoral ageing did not affect the aligner thickness in any of the groups., (© 2024 The Author(s). Orthodontics & Craniofacial Research published by John Wiley & Sons Ltd.)