The existing literature on Mental Workload (MWL) has aimed at understanding processes involved in MWL and their measurements. In addition to subjective and behavioral measures, physiological measurements has shown promising perspectives with respect to workload evaluation. However, their heterogeneity leads to questions their reliability, dependencies and co-variations. The present study aims at identifying objective psychophysiological markers of standardized MWL associated with varying subjective and behavioral MWL-induced variations. First, we defined pre-standardized taskload levels allowing to assess the stability of physiological markers, across MWL variations. Data was collected under a computer-based controlled laboratory setting (MATB-II) mimicking activities that aircraft pilots perform during flight. Standardization procedures were applied to the MATB-II, allowing the identification of scenarios inducing low, medium and high MWL levels, without modifying the number and type of subtasks involved. Participants’ subjective workload evaluations and behavioral performances followed the taskload manipulation. Then, we identified at least three types of physiological measures (cardiac, ocular and electroencephalographic) affected by taskload, notably discriminating high workload conditions. Correlation analyses showed that cardiac and EEG measures are consistently associated with subjective MWL estimations. These results extend our knowledge of MWL to varying levels of taskload tested in an operational task, while confirming the reliability of psychophysiological markers for the detection of varying MWL. Ultimately, this knowledge is expected to be applied in industries for developing new products, to assess new designs with regards to their impact on users’ workload and performances, throughout the design cycle.