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Background: Anserine bursitis is characterized by the presence of spontaneous pain with tenderness at the inferomedial aspect of the knee joint. Neural prolotherapy aims to relieve pain of a variety of chronic musculoskeletal disorders. The study aim was to explore the short-term efficacy of neural prolotherapy (subcutaneous perineural injection of dextrose 5% solution) versus local corticosteroid injection for pain relief and improvement of function in patients with chronic anserine bursitis. The enrolled patients were randomly assigned to receive neural prolotherapy (subcutaneous perineural injection of dextrose 5% solution) (neural prolotherapy group) or a single local soft tissue injection of corticosteroid (corticosteroid group). Outcome measures included Western Ontario McMasters Universities osteoarthritis index, assessment of overall anserine bursitis pain severity using the visual analogue scale, patient's global assessment of anserine bursitis severity using the visual analogue scale, and clinical assessment for the presence of tenderness on the anserine bursa region. Patients were evaluated before injection and after intervention by 4 weeks. Results: The study included 67 lower limbs from 43 patients with chronic anserine bursitis. No significant differences were found between both treatment groups regarding all assessed parameters at the start of the study. After 4 weeks, within-group analysis showed that there was a statistically significant improvement in Western Ontario McMasters Universities osteoarthritis index and its subscales (P ≤ 0.0001), overall anserine bursitis pain severity (P ≤ 0.0001), and patient's global assessment of anserine bursitis severity (P ≤ 0.0001), as well as there was significant improvement regarding the presence of tenderness at the anserine bursa region in both groups in comparison to the preinjection assessment. At the postinjection assessment, between-group analysis showed that there were no significant differences regarding all assessed outcome parameters. All patients in both groups tolerated the injection procedure and were satisfied with the procedure. There was no significant difference between the two groups regarding patients' satisfaction to the procedure results. Improvement was achieved in 86.4% of patients included in the neural prolotherapy group versus 95.2% of patients included in the corticosteroid group. Conclusions: Neural prolotherapy was effective in relieving pain, improving local tenderness and function in patients with chronic anserine bursitis similar to local corticosteroid injection. Trial registration: ClinicalTrials.gov, registration number: NCT04509440. Registered 12 August 2020—Retrospectively registered, [ABSTRACT FROM AUTHOR]