To test the hypothesis that permeability of the blood-brain barrier (BBB) is altered during migraine attack due to enhanced activation of matrix metalloproteinases (MMPs), we investigated MMP-3, MMP-9 and tissue inhibitor of metalloproteases (TIMP)-1 in the external jugular vein during and outside of migraine attacks in 21 patients with migraine without aura. In addition, we measured plasma levels of several other proteins including MMP-7, -8, -10 and TIMP-2. We used Rules-Based Medicine multi-analyte profiling and protein array technologies to study plasma concentration of MMPs. There was no difference in MMP-9 and TIMP-1 levels between ictal and interictal periods. We found significantly decreased plasma levels of MMP-3 in the external jugular (P = 0.002) and cubital (P = 0.008) vein during attacks compared with outside of attacks. We found no correlation of ictal or interictal MMP-3, MMP-9 and TIMP-1 to migraine duration and frequency analysed in 21 patients (P > 0.05). There was no difference between ictal and interictal plasma levels of MMP-7, -8, -10 and TIMP-2 (P > 0.05). Our data suggest that plasma MMP-9 cannot be used as a biomarker of BBB disruption in migraine without aura. Decreased MMP-3 levels are an interesting and unexpected finding warranting further investigation.