Aims: To test whether manipulation of mechanical pain sensitivity (MPS) of the masseter muscle is reflected in quantitative measures of entropy. Methods: In a randomized, single-blinded, placebo-controlled design, 20 healthy volunteers had glutamate, lidocaine, and isotonic saline injected into the masseter muscle. Self-assessed pain intensity on a numeric rating scale (NRS) was evaluated up to 10 minutes following the injection, and MPS was evaluated after application (at 5 minutes and 30 minutes) of three different forces (0.5 kg, 1 kg, and 2 kg) to 15 different sites of the masseter muscle. Finally, the entropy and center of gravity (COG) of the pain sensitivity scores were calculated. Analysis of variance was used to test differences in means of tested outcomes and Tukey post hoc tests were used to adjust for multiple comparisons. Results: The main findings were: (1) Compared with both lidocaine and isotonic saline, glutamate injections caused an increase in peak, duration, and area under the NRS pain curve (P .01); (2) A pressure of 2 kg caused the highest NRS pain scores (P .03) and entropy values (P .02); (3) Glutamate injections caused increases in entropy values when assessed with 0.5 kg and 1.0 kg but not with 2.0 kg of pressure; and (4) COG coordinates revealed differences between the x coordinates for time (P .01) and time and force for the y coordinates (P .01). Conclusion: These results suggest that manipulation of MPS of the masseter muscle with painful glutamate injections can increase the diversity of MPS, which is reflected in entropy measures. Entropy allows quantification of the diversity of MPS, which may be important in clinical assessment of pain states such as myofascial temporomandibular disorders. Keywords: entropy, mechanical sensitization, musculoskeletal pain, pain assessment, palpation