There is no established technique that directly quantifies lower limb tissue perfusion. Blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) is an MRI technique that can determine skeletal muscle perfusion. BOLD-MRI relies on magnetic differences of oxygenated and deoxygenated hemoglobin, and regional changes in oxy/deoxyhemoglobin ratio can be recorded by T2* weighted MRI sequences. We aimed to test whether BOLD-MRI can differentiate lower limb tissue perfusion in peripheral arterial occlusive disease (PAOD) patients and healthy controls. Twenty-two PAOD patients and ten healthy elderly volunteers underwent lower limb BOLD-MRI. Reactive hyperemia was provoked by transient cuff compression and images of the gastrocnemius and soleus muscles were continuously acquired at rest, during ischemia and reperfusion. Key BOLD parameters were baseline T2* absolute value and time to T2* peak value after cuff deflation (TTP). Correlations between imaging parameters and ankle-brachial index (ABI) was investigated. The mean TTP was considerably prolonged in PAOD patients compared to healthy controls (m. gastrocnemius: 111 ± 46 versus 48 ± 22 s, = 0.000253; m. soleus: 100 ± 42 versus 41 ± 30 s, = 0.000216). Both gastrocnemius and soleus TTP values correlated strongly with ABI (-0.82 and -0.78, < 0.01). BOLD-MRI during reactive hyperemia differentiated most PAOD patients from healthy controls. TTP was the most decisive parameter and strongly correlated with the ABI.