There have been many studies attempting to disentangle the relation between motivation and mental fatigue. Mental fatigue occurs after performing a demanding task for a prolonged time, and many studies have suggested that motivation can counteract the negative effects of mental fatigue on task performance. To complicate matters, most mental fatigue studies looked exclusively at the effects of extrinsic motivation but not intrinsic motivation. Individuals are said to be extrinsically motivated when they perform a task to attain rewards and avoid punishments, while they are said to be intrinsically motivated when they do for the pleasure of doing the activity. To assess whether intrinsic motivation has similar effects as extrinsic motivation, we conducted an experiment using subjective, performance, and physiological measures (heart rate variability and pupillometry). In this experiment, 28 participants solved Sudoku puzzles on a computer for three hours, with a cat video playing in the corner of the screen. The experiment consisted of 14 blocks with two alternating conditions: low intrinsic motivation and high intrinsic motivation. The main results showed that irrespective of condition, participants reported becoming fatigued over time. They performed better, invested more mental effort physiologically, and were less distracted in high-level than in low-level motivation blocks. The results suggest that similarly to extrinsic motivation, time-on-task effects are modulated by the level of intrinsic motivation: With high intrinsic motivation, people can maintain their performance over time as they seem willing to invest more effort as time progresses than in low intrinsic motivation.