Objectives: For experimental evaluation of sequential aligner systems, the conventional sequence of aligner thickness (0.5 to 0.8 mm) and a novel sequence using thinner 0.4- and 0.3-mm films were investigated biomechanically in four mechanical in-vitro studies. Materials and methods: The first two studies focused on determining mechanical loads exerted during labio-palatal translation of a maxillary central incisor and derotation of a mandibular canine when using aligners with conventional thicknesses of 0.5 to 0.75 mm. The third and fourth studies examined the mechanical load during different movements of a maxillary central incisor by use of 0.3- and 0.4-mm novel, thinner aligners. All experiments were performed on specifically designed test setups equipped with acrylic casts from which the corresponding measurement tooth was separated and mounted on a three-dimensional (3D) movement unit via a 3D force-moment (F/M) sensor. Results: Even for small movements of 0.5 mm and 10 degrees, quite high F/M values, up to 8.4 N and 89.2 Nmm, respectively, were recorded for the 0.5-mm aligner. Use of aligners thermoformed from novel 0.4-mm film resulted in mean reduction in force and moment of 35% and 45%, respectively, for translation and rotation of the maxillary right central incisor (tooth 11). Aligners fabricated from 0.3-mm film showed insufficient form stability during repeated seating and removal from the test cast, and should therefore also be unsuitable for clinical application. Conclusions: When using the thinnest 0.5-mm film, the risk of overloading the periodontal structures is quite high. Based on these in-vitro experiments, a novel aligner thickness sequence is recommended using 0.4-, 0.5- and 0.75-mm aligners. This sequence substantially reduces the F/M values exerted by the first and thinnest aligner and ensures relatively constant load increases for each individual setup step. Keywords: aligner sequence, aligner thickness, force, orthodontic aligner, overloading