Monitoring pilots’ cognitive states becomes increasingly important in aviation. Physiological measurement can detect increased mental workload (MWL) even before performance declines. Yet, changes in MWL are rarely varied systematically and few studies control for confounding effects of other cognitive states. The present study targets these shortcomings by analysing the effects of stepwise increased MWL on cortical activation, while controlling for mental fatigue (MF). 35 participants conducted a simulated flight with an incorporated adapted n-back and monitoring task. We recorded cortical activation with concurrent EEG and fNIRS measurement, performance, self-reported MWL and MF. Our results show the successful manipulation of MWL without confounding effects of MF. Higher task difficulty elicited higher subjective MWL ratings, performance decline, higher frontal theta activity and reduced frontal deoxyhaemoglobin (Hbr) concentration. Using both EEG and fNIRS, we could discriminate all induced MWL levels. fNIRS was more sensitive to tasks with low difficulty, and EEG to tasks with high difficulty. Our findings further suggest a plateau effect for high MWL that could present an upper boundary to individual cognitive capacity. Our results highlight the benefits of physiological measurement in aviation, both for assessment of cognitive states and as a data source for adaptive assistance systems.