Myofascial pain associated with myofascial trigger points (MTrPs) is a common cause of nonarticular 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 subnanogram level of concentration. The system includes a microdialysis needle capable of continuously collecting extremely small samples (approximately 0.5 microl) of physiological saline after exposure to the internal tissue milieu across a 105-microm-thick semi-permeable membrane. This membrane is positioned 200 microm from the tip of the needle and permits solutes of <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 microanalytical laboratory, employing immunoaffinity capillary electrophoresis and capillary electrochromatography, performed analysis of selected analytes. Concentrations of protons, bradykinin, calcitonin gene-related peptide, substance P, tumor necrosis factor-alpha, interleukin-1beta, serotonin, and norepinephrine were found to be significantly higher in the active group than either of the other two groups (P < 0.01). pH was significantly lower in the active group than the other two groups (P < 0.03). In 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.