Objective: Physical exercise decreases disability and pain associated with chronic articular cartilage degradation. However, understanding of the pathology is lacking. In this study, the levels of 17 biomarkers of inflammation and cartilage degradation were measured in synovial fluid (SF) before and after a 30-minute run in able-bodied and previously-injured individuals. Materials & Methods: Four able-bodied recreational runners (3 men and 1 woman: 24 ± 2 years, 68 ± 7 kg, and 173 ± 9 cm) and 4 recreational runners who had undergone a unilateral anterior cruciate ligament reconstruction (ACLr) (2 men and 2 women: 23 ± 1 years, 71 ± 6 kg, and 175 ± 4 cm) were recruited to participate in this study. Using a saline-assisted method, SF was aspirated before and after both a 30-minute unloading and 30-minute exercise session. Samples were corrected for blood contamination and analyzed for 15 cytokines and 2 matrix metalloproteinases (MMPs). Mixed model analyses were used to determine the main effects of session, case/control status, pre/post aspirations, and the interactions between case/control status and pre/post aspirations. Results: Blood protein contamination was calculated and accounted for in 15 of 32 synovial fluid samples. Granulocyte colony stimulating factor (GCSF) was the only detectable cytokine of the 15 analyzed. No statistical differences were found in GCSF concentrations between pretreatment and posttreatment aspirations (p = 0.45), ACLr and able-bodied control groups (p = 0.60), or unloading and exercise sessions (p = 0.96). MMP-13 was undetectable. No statistical differences were found in MMP-3 between pretreatment and posttreatment aspirations (p = 0.15), ACLr and able-bodied control groups (p = 0.85), or unloading and exercise sessions (p = 0.14).Conclusions: Two (GCSF and MMP-3) of the 17 measured biomarkers were detectable. There were no significant differences in either GCSF or MMP-3 due to a 30-minute run or 30-minute unloading period in either the able-bodied or ACLr participants. Further, there were no significant differences between biomarker concentrations and case-control status. A novel method of controlling for blood contamination in synovial fluid samples was implemented.