Objective: To investigate the relationship between the existence of occlusal interference and masticatory muscle hyperalgesia by exploring the stimulus-response relationship between the duration of occlusal interference and masticatory muscle mechanical withdrawal threshold., Methods: Occlusal interference with 0.4 mm-thick crowns on rat molars was removed under anaesthesia at 2, 3, 4, 5, and 6 d after wear, and masticatory muscle mechanical withdrawal threshold was tested at 1, 3, 5, 7, 10, 14, 21 and 28 d., Results: Decreased mechanical withdrawal thresholds were detected in temporal muscles and masseter muscles on both sides following occlusal interference (P < 0.05). After removal of crowns at 2, 3, 4 and 5 d, rats exhibited the similar head withdrawal thresholds as the sham-application control in masticatory muscles on both sides at 10, 14, 14 and 21 d (P > 0.05). No significant differences were detected between the contralateral side with the ipsilateral side (P < 0.05). After removal of crowns at 6 d, rats still exhibited significantly decreased head withdrawal thresholds[right temporal muscle: (1.365±0.018) N; right masseter: (1.437 ± 0.024) N] in masticatory muscles on both sides until the last day of the experiment[the sham-application control: right temporal muscle: (1.554±0.040) N, P < 0.001; right masseter: (1.546±0.019) N, P < 0.001]., Conclusions: The mechanical hyperalgesia can disappear after removal of the occlusal interference at 5 d, and the existence of the occlusal interference is positively correlated with the duration of the mechanical hyperalgesia.