SUMMARY This controlled longitudinal intervention study investigated changes in cytokines and biomarkers of bone and tissue metabolism within gingival crevicular fluid (GCF) from patients undergoing orthodontic treatment. GCF was collected on Periopaper™ strips (Oraflow Inc., USA) from 20 volunteers at baseline, before tooth extraction and appliance placement and then at intervals during orthodontic treatment. Samples were taken 10 weeks following first appliance placement: at four hours; 7 days; and 42 days after application of distalising forces to maxillary canine teeth. Cytokines (GM-CSF, interferon-gamma, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNFalpha), tissue biomarkers (MMP-9, TIMP-1 & 2) and bone metabolism indicators (RANKL and OPG) were measured in GCF using multiplex assays. Tension sites adjacent to canines showed significant increases in IL-1beta, IL-8, TNFalpha, MMP-9 and TIMPs 1 and 2 across all time points following force application, while compression sites exhibited increases in IL-1beta and IL-8 after 4 hours, MMP-9 after 7 and 42 days and RANKL after 42 days. These data demonstrate that high levels of pro-inflammatory cytokines and biomarkers of tissue and bone metabolism in GCF are associated with orthodontic force application. Elevated levels were evident at 4 hours but continued for periods of up to 6 weeks. The data suggest that GCF biomarker analysis may help optimise orthodontic forces for individual patients.