Objectives: Despite the broad use of occlusal splints in the treatment of temporomandibular disorders, the efficacy of splints is a source of controversy. In this study, the effectiveness of occlusal splints on the electromyographic activity of the masseter and temporal muscles in healthy individuals was investigated. Method and Materials: An occlusal splint was made for 25 healthy individuals. Surface electromyographic recordings were done during maximum clenching without the splint and immediately after application of the splint. The relative level of activity in the masseter and temporal muscles was quantified by means of activity index. Paired t test and Wilcoxon signed-rank tests were used for data analysis. Results: The mean electromyographic activities of masseter and temporal muscles (in mV/s) were 0.7712 and 1.0488 without the splint and 0.8396 and 0.9276 immediately after application of the splint. There was no significant difference between the electromyographic activities of both muscles before and after insertion of the splints. The activity index increased after application of the occlusal splints. This increase was also insignificant. Conclusion: Immediate application of occlusal splints has no significant effect on the activity of masseter and temporal muscles. It could decrease the relative activity of the temporal to the masseter muscle (increasing the activity index). Keywords: electromyography, masseter physiology, maximum clenching, muscle physiology, occlusal splint, temporal physiology