Background and purpose Bone healing is a complex process influenced by growth factors, cytokines, and other mediators. The regulation of this process is not well understood. In this pilot study, we used microdialysis technology in a critical-size bone defect in rat femurs to determine the feasibility of measuring cytokines and growth factors in the first 24 h after injury. Methods A 5-mm defect, stabilized by a plate, was created in the femurs of 30 male Wistar rats. The microdialysis probe (with 100 kDa molecular weight cutoff) was inserted into the defect and microdialysates were collected continuously for up to 24 h. Total protein concentration, interleukin-6 (IL-6) concentration, and transforming growth factor-β1 (TGF-β1) concentration were assessed under different conditions. Results Microdialysis allowed continuous and consistent protein collection over 24 h from a critical-size bone defect starting at the time of injury. IL-6 was secreted within the first 3 h after the injury. The highest IL-6 concentration (344 pg/mL) was measured between 12 and 15 h after surgery. Addition of bovine serum albumin to the perfusate resulted in detectable concentrations of TGF-β1 ranging from 10 to 23 pg/mL. Interpretation Continuous sampling over 24 h of proteins from a bone defect directly after the injury is feasible and provides the opportunity for a detailed analysis of the initial stages of bone healing.