Recent studies about learning and instruction use cognitive load measurement to pay attention to the human cognitive resources and to the consumption of these resources during the learning process. In order to validate different measures of cognitive load for different cognitive load factors, the present study compares three different methods of objective cognitive load measurement and one subjective method. An experimental three-group design (N = 78) was used, with exposure to seductive details (extraneous cognitive load factor), mental animation tasks (germane cognitive load factor), or the basic learning instruction (control group). Cognitive load was measured by the rhythm method (Park and Brünken 2015), the index of cognitive activity (ICA) (Marshall 2007), and the subjective ratings of mental effort and task difficulty (Paas 1992). Eye-tracking data were used to analyze the attention allocation and as an indicator for cognitive activity. The results show a significantly higher cognitive load for the mental animation group in contrast to the control and the seductive detail group, indicated by rhythm method and subjective ratings, as well as a higher cognitive activity, indicated by eye tracking. Furthermore, the mental animation group shows significantly higher comprehension performance in contrast to the seductive detail group and significantly higher transfer performance in contrast to the control group. The ICA values showed no significant differences in cognitive load. The results provide evidence for the benefits of combining eye-tracking analysis and the results of cognitive load ratings or secondary task performance for a direct and continuous cognitive load assessment and for a differentiating access to the single cognitive load factors.