Background <p>Considering the effectiveness of platelet-rich plasma (PRP) in the treatment of knee osteoarthritis and the existence of different methods of preparing PRP, it is necessary to investigate various techniques as well as the number of leukocytes and platelets in the solutions prepared with these techniques and their effectiveness.</p> Purpose <p>This study aimed to compare the effectiveness of PRP obtained through single centrifuged or double centrifuged methods, as well as a placebo injection, in addressing pain, range of motion, and physical performance in patients with knee osteoarthritis.</p> Study design <p>This study was a double-blind, randomized Controlled clinical trial.</p> Methods <p>A randomized controlled trial enrolled 45 participants aged 50 to 75 years diagnosed with knee osteoarthritis, in the years 2021–2022, at the Sports and exercise Medicine Clinic. The participants were randomly assigned to one of three groups: platelet-rich plasma injection, single centrifuged PRP, double centrifuged PRP, or normal saline as a placebo. Pain intensity, knee range of motion, functional tests, and WOMAC Questionnaire scores were evaluated before and after one, three, and six months of the intervention. Statistical analyses were employed to assess the data and identify any superior group post-intervention and during the follow-up period.</p> Results <p>The findings of this investigation demonstrated notable variations among the groups in the ROM 122.14 ± 6.71 (<i>p</i> = 0.001) and Stair-up 25.21 ± 9.45 (<i>p</i> = 0.014) variables one month after the intervention in double-centrifuged group and ROM 118.92 ± 11.95 (<i>p</i> = 0.001) in single-centrifuged group compared to the control group. Furthermore, there was significant improvement (<i>p</i> &lt; 0.05) in the majority of variables within the single and double centrifuged groups compared to the control group at three and six-months post-intervention. Nevertheless, no significant differences were observed in any of the variables between the single and double centrifuged groups except the Stair-Up test after six-month post-intervention.</p> Conclusion <p>Platelet-rich plasma (PRP) exhibits the capacity to enhance pain relief, increase range of motion, and ameliorate function and WOMAC scores in knee osteoarthritis. Both single and double centrifuged PRP methodologies yield comparable effects, but double centrifugation yields more prompt positive outcomes. Consequently, both techniques are viable options in the PRP injection protocol for treating knee osteoarthritis.</p> Clinical trial registration <p>Clinical trial registration: IRCT20140907019073N5.</p>