Background: Migraine is a disabling chronic episodic disorder. Attack frequency progressively increases in some patients. Incremental cortical excitability has been implicated as a mechanism underlying progression. Cortical spreading depression (CSD) is the electrophysiological event underlying migraine aura, and a headache trigger. We hypothesized that CSD events during frequent migraine attacks condition the cortex to increase the susceptibility to further attacks. Methods: A single daily CSD was induced for 1 or 2 weeks in mouse frontal cortex; contralateral hemisphere served as sham control. At the end of CSD conditioning, occipital CSD susceptibility was determined by measuring the frequency of CSDs evoked by topical KCl application. Results: Sham hemispheres developed 8.4 ± 0.3 CSDs/hour, and did not significantly differ from naïve controls without prior cranial surgery (9.3 ± 0.4 CSDs/hour). After 2 but not 1 week of daily CSD conditioning, CSD frequency (4.9 ± 0.3 CSDs/hour) as well as the duration and propagation speed were reduced significantly in the conditioned hemispheres. Histopathological examination revealed marked reactive astrocytosis without neuronal injury throughout the conditioned cortex after 2 weeks, temporally associated with CSD susceptibility. Conclusions: These data do not support the hypothesis that frequent migraine attacks predispose the brain to further attacks by enhancing tissue susceptibility to CSD.