Purpose: The mouthpiece is the standard interface for spirometry tests. Although the use of a mouthpiece can be challenging for patients with orofacial weakness, maintaining a proper seal with a facemask can be an issue for healthy individuals during forceful efforts. We compared respiratory muscle activity and tests using a mouthpiece and facemask in healthy adults to investigate whether they can be used interchangeably. Methods: In this observational study, subjects (n 5 12) completed forced vital capacity, maximal respiratory pressure, and peak cough flow with a mouthpiece and facemask. Root mean square values of the genioglossus, diaphragm, scalene, and sternocleidomastoid were compared between conditions. Results: When switching from a mouthpiece to a facemask, significantly higher values were seen for peak cough flow (average bias 5 254.36 L/min, P , .05) and the difference seen with maximal expiratory pressure (MEP) and maximal inspiratory pressure (MIP) were clinically significant (average bias: MEP 5 27.33, MIP 5 25.2). In addition, the submental activity was significantly greater when MIP was conducted with a mouthpiece. No significant differences were seen in respiratory muscle activity during resting breathing or spirometry. Conclusions: There are clinically significant differences with cough and MEP tests, and neck muscles are activated differently based on interface. Considering the small sample size, our findings suggest a facemask may be used to complete some pulmonary function tests.