AbstractIn light of the educational challenges brought about by the COVID-19 pandemic, there is a growing need to bolster online science teaching and learning by incorporating evidence-based pedagogical principles of Learning Experience Design (LXD). As a response to this, we conducted a quasi-experimental, design-based research study involving nN = 183 undergraduate students enrolled across two online classes in an upper-division course on Ecology and Evolutionary Biology at a large R1 public university. The study extended over a period of 10 weeks, during which half of the students encountered low-stakes questions immediately embedded within the video player, while the remaining half received the same low-stakes questions after viewing all the instructional videos within the unit. Consequently, this study experimentally manipulated the timing of the questions across the two class conditions. These questions functioned as opportunities for low-stakes content practice and retention, designed to encourage learners to experience testing effect and augment the formation of their conceptual understanding. Across both conditions, we assessed potential differences in total weekly quiz grades, page views, and course participation among students who encountered embedded video questions. We also assessed students’ self-report engagement, self-regulation, and critical thinking. On average, the outcomes indicated that learners exposed to immediate low-stakes questioning exhibited notably superior summative quiz scores, increased page views, and enhanced participation in the course. Additionally, those who experienced immediate questioning demonstrated heightened levels of online engagement, self-regulation, and critical thinking. Moreover, our analysis delved into the intricate interplay between treatment conditions, learners’ self-regulation, critical thinking, and quiz grades through a multiple regression model. Notably, the interaction between those in the immediate questioning condition and self-regulation emerged as a significant factor, suggesting that the influence of immediate questioning on quiz grades varies based on learners’ self-regulation abilities. Collectively, these findings highlight the substantial positive effects of immediate questioning of online video lectures on both academic performance and cognitive skills within an online learning context. This discussion delves into the potential implications for institutions to continually refine their approach in order to effectively promote successful online science teaching and learning, drawing from the foundations of pedagogical learning experience design paradigms and the testing effect model.