Background Cognitive overload can inhibit learning, and cognitive load theory-based instructional design principles can be used to optimize learning situations. This study aims to investigate the effect of implementing cognitive load theory-based design principles in virtual reality simulation training of mastoidectomy. Methods Eighteen novice medical students received 1 h of self-directed virtual reality simulation training of the mastoidectomy procedure randomized for standard instructions (control) or cognitive load theory-based instructions with a worked example followed by a problem completion exercise (intervention). Participants then completed two post-training virtual procedures for assessment and comparison. Cognitive load during the post-training procedures was estimated by reaction time testing on an integrated secondary task. Final-product analysis by two blinded expert raters was used to assess the virtual mastoidectomy performances. Results Participants in the intervention group had a significantly increased cognitive load during the post-training procedures compared with the control group (52 vs. 41 %, p = 0.02). This was also reflected in the final-product performance: the intervention group had a significantly lower final-product score than the control group (13.0 vs. 15.4, p < 0.005). Conclusions Initial instruction using worked examples followed by a problem completion exercise did not reduce the cognitive load or improve the performance of the following procedures in novices. Increased cognitive load when part tasks needed to be integrated in the post-training procedures could be a possible explanation for this. Other instructional designs and methods are needed to lower the cognitive load and improve the performance in virtual reality surgical simulation training of novices.