The objectives of this study were to investigate the structure of Invisalign appliances (Align Technology, Santa Clara, Calif) after intraoral exposure, and to qualitatively and quantitatively characterize the substances leached from the aligners after accelerated in vitro aging. Samples of Invisalign appliances were randomly selected from 10 patients before intraoral placement and after retrieval, and the prepared specimens were subjected to (1) bright-field optical reflection microscopy to study the surface morphology; (2) Fourier transform infrared microspectroscopy to characterize the in vivo changes in molecular composition induced on appliance surfaces, (3) scanning electron microscopy and energy dispersive X-ray microanalysis to identify the elemental composition of integuments formed on the surface, and (4) Vickers hardness (HV200) testing. Another set of reference and retrieved appliances was subjected to artificial aging for 2 weeks, and the extracts were subjected to gas chromatography-mass spectroscopy. The retrieved appliances demonstrated substantial morphological variation relative to the as-received specimens involving abrasion at the cusp tips, adsorption of integuments, and localized calcification of the precipitated biofilm at stagnation sites. Buccal segments of retrieved appliances showed an increase in hardness, which might be attributed to mastication-induced cold work; however, the clinical implication of this effect on mechanotherapy is unknown. In vitro aged and retrieved appliances were found to leach no traceable amount of substances in an ethanol aging solution.