Myofascial pain associated with myofascial trigger points (MTrPs) is a common cause of non-articular musculoskeletal pain. Although the presence of MTrPs can be determined by soft tissue palpation, little is known about the mechanisms and biochemical milieu associated with persistent muscle pain. A microanalytical system was developed to measure the in-vivo biochemical milieu of muscle in near real time at the sub-nanogram level of concentration, the system including a microdialysis needle capable of continuously collecting extremely small samples (~ 0.5 µl) of physiological saline after exposure to the internal tissue milieu across a 105-µm thick semi-permeable membrane. This membrane is positioned 200 µm from the tip of the needle and permits solutes of less than 75 kDa to diffuse across it. Three subjects were selected from each of three groups (total 9 subjects): Normal (no neck pain, no MTrP); Latent (no neck pain, MTrP present); Active (neck pain, MTrP present). The microdialysis needle was inserted in a standardized location in the upper trapezius muscle. Due to the extremely small sample size collected by the microdialysis system, an established micro-analytical laboratory, employing immunoaffinity capillary electrophoresis (ICE) and capillary electrochromatography (CEC), performed analysis of selected analytes. Concentrations of protons (H⁺), bradykinin, calcitonin gene-related peptide, substance P, tumor necrosis factor- , Interleukin -1, serotonin, and norepinephrine were found to be significantly higher in the Active group than either of the other two groups (p<.01). pH was significantly lower in the Active group than the other two groups (p<.03). Conclusion: The described microanalytical technique enables continuous sampling of extremely small quantities of substances directly from soft tissue, with minimal system perturbation and without harmful effects on subjects. The measured levels of analytes can be used to distinguish clinically distinct groups.