Transcranial magnetic stimulation (TMS) has been widely used in human cognitive neuroscience to examine the causal role of distinct cortical areas in perceptual, cognitive and motor functions. However, it is widely acknowledged that the effects of focal cortical stimulation on behaviour can vary substantially between participants and even from trial to trial within individuals. Here we asked whether spontaneous fluctuations in alertness can account for the variability in behavioural and neurophysiological responses to TMS. We combined single-pulse TMS with neural recording via electroencephalography (EEG) to quantify changes in motor and cortical reactivity with fluctuating levels of alertness defined objectively on the basis of ongoing brain activity. We observed rapid, non-linear changes in TMS-evoked neural responses – specifically, motor evoked potentials and TMS-evoked cortical potentials – as EEG activity indicated decreasing levels of alertness, even while participants remained awake and responsive in the behavioural task.IMPACT STATEMENTA substantial proportion of inter-trial variability in neurophysiological responses to TMS is due to spontaneous fluctuations in alertness, which should be controlled for during experimental and clinical applications of TMS.