Mental fatigue is a transient psychophysiological state characterized by impaired cognition and behavior across a range of dynamic contexts. Despite increasing interest in this phenomenon, its (neuro)physiologic representations remain unclear. This systematic review aimed to quantify the range of (neuro)physiologic outcomes and methodologies used to investigate mental fatigue in laboratory-based settings. Across the 72 studies meeting our inclusion criteria, we identified 30 unique physiologic, four visual outcomes, and the application of several neuroimaging techniques investigating neuronal function. Mental fatigue increased heart rate, systolic and diastolic blood pressure, mean arterial pressure, low frequency, and root mean square of successive differences (RMSSD), and reduced standard deviation of normal-to-normal intervals (SDNN) (all P ≤ 0.04) when compared with controls. Applying electroencephalography to investigate delta, theta, and alpha bandwidths may provide useful insights into this phenomenon, and functional near-infra-red spectroscopy to right-lateralized frontoparietal regions would be helpful to investigate cortical activity change in response to mental fatigue. More data are needed across a range of methodological contexts in order to further determine the (neuro)physiological manifestations of mental fatigue. However, this review provides direction to researchers and will assist them in navigating and considering the range of options available.