The aim of this study was to analyze the effect of occlusal modifications on the muscular activity of the masseter and anterior temporalis muscles. The study included 41 healthy dentate subjects who were examined in relation to the muscle activity of the masseter and anterior temporalis muscles recorded by surface electromyography (EMG) bilaterally in two different sessions. Occlusal plastic strips (thickness: 0.4 or 0.8 mm) were placed on different mandibular teeth to simulate different bite constellations (unilateral, bilateral transversal, and bilateral diagonal). Controlled by visual feedback, the subjects performed submaximum occlusion at 10% and 35% of maximum voluntary contraction (MVC). The activity ratios of the muscles were analyzed by two-way repeated measurement analysis of variance (ANOVA), and the reliability of muscle activity data was determined by intraclass correlation coefficient (ICC) analysis. The activity ratios of the masseter muscles were not significantly different under various biting conditions. In contrast, the anterior temporalis muscles showed significant differences (P < 0.001) between unilateral configurations and the other biting conditions (bilateral transversal or diagonal), in particular during biting at 10% MVC. In general, ICC values revealed low to moderate reliability of the measurements of muscle activity. Under controlled submaximum occlusal loading, the activity behavior of the masseter muscles remained stable, whereas the anterior temporalis muscles reacted differently to distinct occlusal biting configurations. The results support the assumption that the anterior temporalis muscles might operate as fine-tuning muscles when asymmetric bite force distributions occur, for instance during chewing, caused by food fragments between the teeth.