Temporomandibular joint (TMJ) pain is a predominant sign and symptom in patients with temporomandibular disorder, and a common cause of chronic orofacial pain. Arthrocentesis of the upper joint space proved to be effective in reducing TMJ-related pain and reestablishing normal mandibular range of movement in patients diagnosed for a ‘closed lock’. Using the therapeutic approach of arthrocentesis in TMJ-related instances of capsulitis/synoyitis (C/S) with a recency of first pain onset of ≤6 months, the purpose of the present study was to evaluate whether the TMJ-related variable synovial fluid (SF) level of TNF- α may be linked to the cessation of related signs and symptoms associated with the performance of arthrocentesis and hydraulic distension. In 23 patients with a specific temporomandibular disorder diagnosis of unilateral C/S with a recency of first pain onset of ≤6 months, TMJ SF aspirates were obtained from the pain and contralateral non-pain sides immediately before and after arthrocentesis. Visual analog scales were used for pre- and postoperative self-assessment of TMJ-related pain during function, while enzyme-linked immunosorbent assays were applied for measurement of the tumor necrosis factor- α (TNF- α) concentration. With a mean SF TNF- α level of 13.91 ng/ml associated with the pain side, and a mean SF TNF- α level of 7.73 ng/ml associated with the non-pain side, a statistically significant difference was found between the sample groups ( P=0.001). Arthrocentesis led to a significant intraoperative decrease of the respective preoperative SF TNF- α levels, namely 61.64% ( P=0.000) on the pain side and 89.50% ( P=0.000) on the non-pain side, while reduction of TMJ-related pain during function was 73.17% ( P=0.000). Clinical evaluation showed a significant reduction in the prevalence of TMJ-related diagnoses of C/S ( P<0.001). There was no change in the prevalence of associated TMJ-related diagnoses of internal derangement. In view of the fact that the described technique of TMJ SF analysis may be suggested as a valuable diagnostic method for the detection of biochemical SF events, the results of this study should encourage research in its potential uses so that it can become established as a reliable diagnostic approach. Further, the findings may support the concept of bilateral arthrocentesis to be effective in the treatment of patients with a unilateral specific TMD diagnosis of non-chronic C/S.