Prolonged wakefulness alters cortical excitability, which is essential for proper brain function and cognition. However, besides prior wakefulness, brain function and cognition are also affected by circadian rhythmicity. Whether the regulation of cognition involves a circadian impact on cortical excitability is unknown. Here, we assessed cortical excitability from scalp EEG-responses to transcranial magnetic stimulation in 22 participants during 29-h of wakefulness under constant conditions. Data reveal robust circadian dynamics of cortical excitability that were strongest in those individuals with highest endocrine markers of circadian amplitude. In addition, the time course of cortical excitability correlated with changes in EEG synchronization and cognitive performance. These results demonstrate that the crucial factor for cortical excitability, and basic brain function in general, is the balance between circadian rhythmicity and sleep need, rather than sleep homeostasis alone. These findings have implications for clinical applications such as noninvasive brain stimulation in neurorehabilitation