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Abstract Background Oral appliances (OAs) are generally designed to displace the mandible anteriorly and downward, to increase the airway patency. The present study aimed to examine the relationship between genioglossus (GG) muscle activity and mandibular position, considering both anterior and vertical displacements during sleep. Methods Seven healthy male adults aged 29.4 ± 1.99 years were evaluated. Maxillary and mandibular OAs were fabricated from 2-mm-thick resin plates with pressure-welding. The activity of the left GG was recorded using two silver ball electrodes attached to the lingual edge of the mandibular OA. Respiratory status and right masseter muscle activity were measured by an airflow sensor and surface electrodes, respectively. Electroencephalography was used to determine the sleep status. Stage 2 (the second stage of sleep) was defined as the state of sleeping. Four test conditions with different mandibular positions (0 and 50% anterior protrusion) and bite openings (4 mm and 12 mm) were examined. Results GG activity in SL4A (4 mm bite opening, 50% protrusion during sleep) and SL12 (12 mm bite opening, 0% protrusion during sleep) were significantly higher than that in SL4 (4 mm bite opening, 0% protrusion during sleep). Respiratory volume did not significantly differ between all test conditions. Conclusion GG activity is influenced not only by anterior protrusion of the mandible but also by vertical displacement during sleep. Thus, when determining the effectiveness of intraoral appliances in the treatment of obstructive sleep apnea, both protrusion and the size of the mandibular opening should be evaluated and taken into account.