The aim of this follow-up study was to evaluate the effects of Pre-Orthodontic Trainer (POT) appliance on the anterior temporal, mental, orbicularis oris, and masseter muscles through electromyography (EMG) evaluations in subjects with Class II Division 1 malocclusion and incompetent lips. Twenty patients (mean age: 9.8 ± 2.2 years) with a Class II Division 1 malocclusion were treated with POT (Myofunctional Research Co., Queensland, Australia). A group of 15 subjects (mean age: 9.2 ±0.9 years) with untreated Class II Division 1 malocclusions was used as a control. EMG recordings of treatment group were taken at the beginning and at the end of the POT therapy (mean treatment period: 7.43 ± 1.06 months). Follow-up records of the control group were taken after eight months of the first records. Recordings were taken during different oral functions: clenching, sucking, and swallowing. Statistical analyses were undertaken with Wilcoxon and Mann–WhitneyU-tests. During the POT treatment, activity of anterior temporal, mental, and masseter muscles was decreased and orbicularis oris activity was increased during clenching and these differences were found statistically significant when compared to control. Orbicularis oris activity during sucking was increased in the treatment group (P < 0.05). In the control group, significant changes were determined for anterior temporal (P < 0.05) and masseter (P < 0.01) muscle at clenching and orbicularis oris (P < 0.05) muscle at swallowing during observation period. Present findings indicated that treatment with POT appliance showed a positive influence on the masticatory and perioral musculature.