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OBJECTIVE To compare effect of two different orthodontic forces on maxillary canine distalization via evaluation of 30 analytes including cytokines, growth factors, and chemokines in gingival crevicular fluid (GCF) obtained from tension and compression sites. DESIGN Longitudinal, split-mouth, randomized controlled trial. METHODS The upper right and left canines were randomly distalized by a continuous force of either 75 or 150 g, in 15 individuals with Class II division 1 malocclusion. GCF samples were obtained from the tension and the pressure sides of each canine at appliance placement (baseline) and after force application at 24 hours and 28 days without reactivation of the coil spring. The protein content of GCF was analysed by a multiplexed immunoassay. The effects of force, side, and time on the analyte levels were assessed by the Brunner-Langer method. OUTCOME The changes of GCF analyte levels from baseline to 24 hours and 28 days. RANDOMIZATION Coin flipping was used for allocation of two forces. BLINDING The participants and periodontist who performed clinical measurements and GCF sampling were blinded to group assignment and interventions (double-blinded trial). RESULTS All patients completed the study. No harm was observed. When compared to baseline, both forces caused significant up-regulation of tumour necrosis factor-α and interleukin (IL)-1RA in the tension and the pressure sides at 28 days (P < 0.05), but not at 24 hours. Although GCF volume was similar between the two force groups over time (P > 0.05), IL-8 and MCP-1 levels in GCF were significantly lower at the pressure sites receiving higher force (150 g) at 24 hours (P < 0.05). LIMITATIONS Although sample size (15 patients, 30 teeth) was adequate according to the initial power calculation, borderline significances may indicate lack of power or large variability among the samples. CONCLUSIONS Although a higher force of 150 g did not result in increased cumulative canine movement or GCF production, selective host mediators were differentially regulated by the magnitude and duration of the force. REGISTRATION AND TRIAL PROTOCOL The trial was registered retrospectively in the U.S. National Institutes of Health Clinical Trials Registry. Full details of trial protocol NCT03555747 are available on request.