Recent literature suggests that when prolonged, motor imagery (MI) induces mental fatigue and negatively impacts subsequent physical exercise. The aim of this study was to confirm this possibility with neurophysiological and self-reported measures. Thirteen participants performed 200 imagined isometric knee extension contractions (Prolonged MI condition) or watched a documentary (Control condition), and then performed 150 actual isometric knee extensions. Electroencephalography was continuously recorded to obtain motor-related cortical potential amplitude at Cz electrode (MRCP, index of motor area activity) for each imagined and actual contraction. Electromyography of the vastus lateralis muscle as well as the perceived effort required to perform prolonged MI, watch the documentary, and perform the actual contractions were measured. During prolonged MI, mental fatigue level, the effort required to imagine the contractions and MRCP amplitude increased over time. The increase in the effort required to imagine the contractions was significantly correlated with the MRCP amplitude. During the physical exercise, a significant condition × time interaction revealed a greater increase over time in perceived effort in the prolonged MI condition compared to the control condition, as well as a specific alteration in EMG RMS of the vastus lateralis muscle. These alterations observed in the presence of mental fatigue during actual contractions, combined with those observed during prolonged MI, suggest that prolonged MI may impair the motor command required to perform imagined or actual contractions. While the observed effect of mental fatigue on MRCP amplitude was clear during MI, future studies should tailor the physical exercise to minimize the exercise-induced decrease in force production capacity and control for its confounding effects on MRCP amplitude in the presence of mental fatigue.