BackgroundKnee osteoarthritis is the most common arthritis. Various treatments such as analgesics, exercise therapy, and surgery in high-grade OA have been shown to reduce pain and improve patients' function; however, determining the optimal treatment remains a challenge. Ozone therapy is one of the injection techniques used for symptom relief in these patients. Therefore, this study aimed to evaluate the effect of ozone injection in mild to moderate knee osteoarthritis. MethodsThirty-three patients with grade II-III knee osteoarthritis based on the Kellgren-Lawrence classification were involved in the study, by block randomisation. Totally 42 knees were included. All patients received exercise therapy, 500 mg of acetaminophen tablets (up to 2 g per day as needed), and healthy nutrition. In a double-blinded method, the intervention group received Ozone injections, but the control group received placebo injections. Functional tests, including timed-up-and-go and 6-min walk tests, were assessed at baseline and immediately after the 6-week intervention. In addition, the pain was measured by VAS score, and stiffness and activity of daily living (ADL) were evaluated by KOOS questionnaire before and after a 6-week intervention and then one and six months afterwards. FindingsImprovements in pain and KOOS scores were seen in both groups in the 6th week of injections (p < 0.05), with significant differences between groups. However, the effects on pain and KOOS scores disappeared in the 1st and 6th months of follow-ups in the control group. Nevertheless, the effects persisted in the intervention group compared to the baseline and control group, which means that in the mentioned time points intervention group showed significant improvement compared to the control group (p < 0.05). In addition, functional tests showed significant differences between the two groups in the 6th week of injections (p < 0.001). InterpretationOzone injection is a non-surgical treatment for mild to moderate knee osteoarthritis that could decrease pain and improve function and ADL of patients in the short to mid-term (3–6 months), so it seems that adding Ozone injection to the routine exercise therapy in management of patients with knee OA could improve outcomes.