The investigation of the physiological and pathological processes involved in fear perception is complicated due to the difficulties in reliably eliciting and measuring the complex construct of fear. This study proposes a novel approach to induce and measure subjective fear and its physiological correlates combining virtual reality (VR) with a mixed-effects model based on skin conductance (SC). Specifically, we developed a new VR scenario applying specific guidelines derived from horror movies and video games. Such a VR environment was used to induce fear in eighteen volunteers in an experimental protocol, including two relaxation scenarios and a neutral virtual environment. The SC signal was acquired throughout the experiment, and after each virtual scenario, the emotional state and fear perception level were assessed using psychometric scales. We statistically evaluated the greatest sympathetic activation induced by the fearful scenario compared to the others, showing significant results for most SC-derived features. Finally, we developed a rigorous mixed-effects model to explain the perceived fear as a function of the SC features. Model-fitting results showed a significant relationship between the fear perception scores and a combination of features extracted from both fast- and slow-varying SC components, proposing a novel solution for a more objective fear assessment.