Background To demonstrate the feasibility of using a susceptibility-based MRI technique with asymmetric spin-echo (ASE) sequence to assess the lower extremity muscle oxygen extraction fraction (OEF) alternations under cuff compression paradigm. Methods Approved by the local institutional human study committee, nine healthy young volunteers participated in this study. All the ASE scans were conducted using a 3 Tesla clinical MRI scanner during resting state (pre), 1-3 min (post1) and 3-5 min (post2) after a pressure of 50 mmHg above individual systolic blood pressure imposed on the thigh. Moreover, near-infrared spectroscopy (NIRS) measurements were performed on the same day under the same cuff compression protocol to verify the accuracy of this susceptibility-based method. Results In all volunteers, the mean MRI based OEF in gastrocnemius (GAS) muscle increased significantly from 0.28 ± 0.02 (pre) to 0.31 ± 0.03 (post1, P < 0.05) and 0.31 ± 0.03 (post2, P < 0.05). In addition, mean OEF in soleus (SOL) muscle went up from 0.31 ± 0.01 (pre) to 0.33 ± 0.03 (post1, P = 0.14) and 0.37 ± 0.04 (post2, P < 0.05). For comparison, NIRS measured 1-%HbO2 (percentage of deoxyhemoglobin concentration within total hemoglobin) in GAS rose significantly from 0.29 ± 0.03 (pre) to 0.31 ± 0.04 (post1, P < 0.05) and 0.31 ± 0.04 (post2, P < 0.05), which confirmed the accuracy of the MRI-based OEF. Conclusion This susceptibility-based OEF quantification technique together with cuff compression paradigm could provide a noninvasive, quantifiable and effective tool for measuring skeletal muscle oxygenation.