Objective: The aim of this study was to determine the relationship of high daily monosodium glutamate (MSG) consumption with glutamate concentrations in jaw muscle, saliva, and serum, and muscle pain sensitivity in healthy participants. Methods: A randomized, double-blinded, placebo-controlled study was conducted to investigate the effect of repetitive consumption of high-dose MSG on glutamate concentration in the masseter muscles measured by microdialysis and muscle pain sensitivity. In five contiguous experimental daily sessions, 32 healthy participants drank MSG (150mg/kg) or NaCl (24mg/kg) diluted with a 400mL soda. The concentrations of glutamate before and after the ingestion were assessed in dialysate and plasma samples on the first and last days. Saliva glutamate concentration was assessed every day. Pressure pain threshold, pressure pain tolerance, autonomic parameters (heart rate, systolic and diastolic blood pressures) and reported side effects also were assessed. Results: No significant change was noted in the baseline concentration of glutamate in the masseter muscle, blood, or saliva, but the peak concentration in the masseter muscle increased significantly between day 1 and 5. A statistically significant increase in systolic and diastolic blood pressures after MSG administration was observed, as well as a significantly higher frequency of reports of nausea and headache in the MSG group. No robust effect of MSG on muscle sensitivity was found. Conclusion: Interstitial glutamate concentration in the masseter muscle is not highly disturbed by excessive repetitive intake of MSG in healthy man.