It has been a long-term goal to develop non-invasive methods that can detect critical levels of tissue hypoxia to help in the management of chronic lower limb ischaemia. In the present study, skeletal muscle oxygenation was measured using a new Clark-type TCPO2 [transcutaneous PO2 (partial pressure of O2)]/PCO2 (partial pressure of CO2) monitoring system and optical NIRS (near-infrared spectroscopy) at graded levels of hypoxaemia using a rabbit model (n=6). The TCPO2/PCO2 probe was placed on the shaved hindlimb to record SPO2 (skin PO2) and SPCO2 (skin PCO2) continuously. A pair of NIRS probes were placed on the limb to monitor HbO2 (oxyhaemoglobin) and Hb (deoxyhaemoglobin). Graded hypoxaemia was achieved by stepwise reductions of FiO2 (fraction of inspired O2) from 30% to 6%. Animals were allowed to recover after each episode of hypoxia at an FiO2 of 30% as indicated by normalized arterial blood PO2. There was a significant (P<0.05) decrease in SPO2 with all grades of hypoxaemia and no significant changes in SPCO2. There was a significant (P<0.05) increase in muscle Hb with all grades of hypoxaemia and a significant (P<0.05) decrease in HbO2 when FiO2 was below 15%. A significant correlation was found between the SPO2 and HbO2 (r=0.92, P<0.001) and both were significantly correlated with arterial blood PO2 (P<0.001). The new TCPO2/PCO2 system, in addition to its application for the assessment of conditions such as chronic venous insufficiency where alteration in skin oxygenation occurs solely, also has potential in conditions such as peripheral vascular disease where both skin and muscle oxygenation may be affected.