Background and objectives: The aim of this study is to evaluate mandibular elevator muscles activity and pain on palpation in the early stages of orthodontic treatment with clear aligners using surface electromyography (sEMG). Materials and methods: Surface electromyography (sEMG) activity and pain level on muscle palpation of masseter and anterior temporalis muscles were recorded in a sample of 16 adult subjects (aged 18–32 years; mean 22.5 +/− 3.5 SD) undergoing orthodontic treatment with clear aligners before the treatment (T0), after 1 month of treatment (two clear aligners) (T1), and after 3 months of treatment (T2) (six clear aligners). A chi-square test for nominal data, a Friedman test, and a Wilcoxon-signed rank test as post hoc analysis were applied. Results: No statistically significant differences in muscular pain were observed. At T1, the sEMG activity of masseter muscles at mandibular rest position showed a statistically significant reduction, but after 3 months (T2), the data appeared similar to T0 (p = 0.03 and p = 0.02). Conclusions: During the treatment with clear aligners, subjects could experience an initial reduction in the masseter basal activity after 1 month of treatment. This effect tends to decrease to baseline levels after 3 months of therapy.