Biomarkers of cartilage metabolism are sensitive to changes in the biological and mechanical environment and can indicate early changes in cartilage homeostasis. The purpose of this study was to determine if a daily locomotion replacement program can serve as a countermeasure for changes in cartilage biomarker serum concentration caused by immobilization. Ten healthy male subjects (mean ± 1 standard deviation, age: 29.4±5.9 years; body mass: 77.7±4.1 kg) participated in the cross-over 5 days bed rest study with three interventions: control (CON), standing (STA) and locomotion replacement training (LRT). Serum samples were taken before, during, and after bed rest. Biomarker concentrations were measured using commercial enzyme-linked immunosorbent assays. Cartilage oligomeric matrix protein (COMP) levels after 24hrs of bed rest decreased independently of the intervention (-16.8 to -9.8%) and continued to decreased until 72hrs of bed rest (minimum, -23.2 to -20.6%). LRT and STA did not affect COMP during bed rests (p=0.056) but there was a strong tendency for a slower decrease with LRT (-9.4%) and STA (-11.7%) compared to CON (-16.8%). MMP-3 levels decreased within the first 24hrs of bed rest (CON: -22.3%; STA: -14.7%; LRT: -17%) without intervention effect. Both COMP and MMP-3 levels recovered to baseline levels during the 6 days recovery period. MMP-1, MMP-9 and TNF-alpha levels were not affected by immobilization or intervention. COMP and MMP-3 are mechanosensitive cartilage biomarkers affected by immobilization, and simple interventions such as standing upright or LRT during bed rest cannot prevent these changes. Clinical significance: Simple locomotion interventions cannot prevent cartilage biomarker change during bed rest. This article is protected by copyright. All rights reserved.