Fatigue is a common problem in healthy individuals, but the effects on cognition are poorly understood. The current experimental study investigated the relation between fatigue and cognitive flexibility. Sixty university students were randomly assigned to an experimental group or a control group. The experimental group received a fatigue-inducing session in which they performed cognitively demanding tasks. The control group received non-demanding tasks. After the intervention, both groups performed a switch task with two task rules of unequal difficulty. Both induced fatigue and fatigue state at baseline were evaluated. Difficulties in task switching, irrespective of task rule, were more pronounced in students in both groups who had higher fatigue at baseline. The experimental group responded slower under all conditions. Moreover, the experimental group took longer to switch from the difficult to the easy task rule compared to the opposite direction. These findings suggest that fatigue negatively affects cognitive flexibility in university students.