Prospective cohort study. The inclusion criteria comprised patients older than 18 years who attended the orthodontic department at the University of L'Aquila (L'Aquila, Italy) and requested treatment with clear orthodontic appliance. Only patients with Class I malocclusion with mild (0-3 mm) or moderate (4-6 mm) crowding were included. The exclusion criteria comprised patients who had medical condition that preclude the use of surface EMG (sEMG), patients who had anterior or posterior open bite, patients who had cross bite that include more than one tooth, patients who had edentulous ridges and patients with temporomandibular disorder. Included patients were assessed at three points in time: at the start of treatment (T0), after 3 months (T1), and after 6 months (T2). The patients were instructed during the study to wear each set of aligners (Nuvola; Gruppo Europeo di Ortodonzia S.r.l., Rome, Italy) for 2 weeks and 22 h/d and to remove them while eating and drinking (except for still water). The following two assessments were done for each patient at T0, T1, and T2: surface electromyography (sEMG) evaluation and a T-Scan digital occlusal analysis evaluation. Both evaluations were performed in centric occlusion and with aligners worn. The assessments were done by the same operator. The sEMG evaluation aimed to assess the muscular symmetry and balance. The sEMG evaluation was performed using an instrument that recorded analogical sEMG signal (BTS TMJOINT, Teethan S.p.a., Garbagnate Milanese, Milano, Italy). The patients were instructed to clench as much as possible and to maintain the same level of contraction during the test. The software automatically selected the 3 s with the most stable sEMG signal. The following outcomes were assessed using the sEMG: 1. Masseter percentage overlapping coefficient (POC), temporalis POC, and mean POC: which is an index of the symmetrical distribution of sEMG potentials within homologous muscular couples. 2. Torque coefficient (TC): compares the activity of the temporalis muscle to that of the contralateral masseter muscle. 3. Activity index (Ac): compares the activity of the temporalis muscle to that of the homolateral masseter muscle 4. Asymmetry index (Asym): compares the activity of the temporalis and masseter muscles of the right side to those of the left side. The digital occlusal analysis was performed using a 100μ thin, flexible horseshoe-shaped Mylar sensor (Novus HD sensor, Tekscan, Inc. S. Boston, MA, USA) that is used to analyze the dynamic and real-time distribution and timing of occlusal contacts and can reproduce 256 levels of varying occlusal force. A first recording was made without aligners, then a second with the aligners worn on the dental arches. The following outcomes were assessed using the digital occlusal analysis: 1. Position of the center of occlusal force (COF). 2. The maximal intercuspation time (MIC) (i.e., the time interval expressed in seconds between the first dental contact and the maximal intercuspation). The null hypothesis was that no difference exists for the sEMG indexes between the 3-time points and the two occlusal conditions. Twenty-six female adult patients (mean age, 33.67 ± 13.33 years) were enrolled in this study. Statistically significant differences were observed in terms of POC for the temporalis and masseter muscles, as well as the mean POC across the three time points and between occlusal conditions (with or without aligner). Hence, null hypothesis was rejected. On the other hand, TC, Ac, Asym, and MIC variables did not exhibit any statistically significant differences. This confirms that the stable positioning of COF on the transverse plane was accompanied by the absence of torquing muscular couples or imbalances in muscular activation. Symmetry in muscular couples' activation (indicated by POC value) remained consistent over the 6-month follow-up period for the occlusion with aligners, and in fact, demonstrated some improvements. In contrast, there was a decline in POC over time during centric occlusion. Statistically significant variation in COF position was observed in the sagittal plane, but not in the transverse plane. This shift in COF position coincided with changes in muscular balance as assessed by surface electromyography. Regarding the anteroposterior position of the COF, an overall slight posterior shift was observed when aligners were worn. After 3 months of full-time aligner usage, an anterior COF position was detectable in centric occlusion, which exhibited statistical significance when compared with the occlusion involving aligners at T0 and T1. The use of clear aligners led to an anterior displacement of the COF during biting in centric occlusion, along with a posterior shift while the aligners were worn in healthy female subjects over a 6-month monitoring period. No significant asymmetries in the COF position on the transverse plane were observed. The alterations in occlusal contact were subsequently followed by a short-term improvement in muscular balance when aligners were used, but a worsening muscular balance over time in centric occlusion condition.