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Due to the increasing usage of virtual learning environments (VLE) in science, it is important to study the factors that contribute to learning. This study explores student-level factors that influence learning outcomes for middle school students in a VLE, Betty's Brain. The analyses used Ordinary Least Squares (OLS) regression to assess the impact of students' perception of pedagogical agents (PA), prior knowledge, learning content difficulty, and anxiety. The findings suggest that prior knowledge, correct actions in the system, and the perception of the helpfulness of pedagogical agents positively influence learning outcomes. Anxiety, however, was found to have a negative impact. This research sheds light on key aspects for optimizing student performance, and informing the design of PA in VLEs.