To examine the implications of the transition from face-to-face to online learning from a psychobiological perspective, this study investigated potential differences in physiological stress parameters of students engaged in online or face-to-face learning and determined whether these can be identified as possible mediators between learning experience and achievement emotions. In a randomized experimental field study, medical students (n = 82) attended either regular face-to-face classes of the microscopic anatomy course or the same practical course online using Zoom videoconferencing platform. The present study investigated Heart Rate Variability (HRV) and salivary cortisol concentration as stress correlates, within the contexts of online and face-to-face learning and compared these parameters with a control group that was measured at rest. Additionally, participants completed a standardized questionnaire about their experienced emotions in relation to task achievement and subjective stress levels. A significant reduction in HRV was found in face-to-face learning, suggesting stronger stress responses in the face-to-face learning environment ([eta][superscript 2] = 0.421, P < 0.001). Furthermore, participants engaged in face-to-face learning showed significantly higher cortisol concentrations ([eta][superscript 2] = 0.115, P = 0.032). Additionally, increased sympathetic activation correlated with the discrete positive emotion of enjoyment exclusively within the face-to-face condition (r = 0.365, P = 0.043). These results indicate that the transfer of a face-to-face practical course in microscopic anatomy to an online learning environment is associated with decreased sympathetic and enhanced vagal cardiovascular influences, together with lower cortisol concentrations in healthy medical students.