Chronic sleep disruption and shift work elevate the risk of neurodegeneration and Alzheimer's disease (AD). While disrupted sleep affects canonical AD biomarkers, its impact on other mechanisms, such as circulating microRNAs (miRNAs), remains less understood. Therefore, we here examined the effects of overnight wakefulness on plasma levels of several miRNAs implicated in neurodegeneration and AD, as well as in sleep and circadian regulation-namely miR-127-3p, miR-132-3p, and miR-142-3p. Following a baseline period in each highly controlled in-lab session, in total 15 healthy normal-weight young men underwent two conditions on separate occasions, in randomised order: a night of normal sleep, and a night of sustained wakefulness. After overnight wakefulness, morning plasma levels of miR-127-3p and miR-142-3p were significantly elevated compared with post-sleep levels. These changes were not associated with the significant increase in self-reported morning stress levels observed after wakefulness compared with sleep. This study is the first to demonstrate that a single night of wakefulness-mimicking overnight shift work-significantly elevates circulating levels of miR-127-3p and miR-142-3p in humans. These findings, though based on a limited sample size, suggest a potential molecular link between sleep loss and neurodegeneration, warranting further investigation. Trial Registration: Clinical Trial number: NCT01800253; www.clinicaltrials.gov.