While adolescents and adults should limit high levels of sedentary behavior, university students spend large amounts of time on sedentary activities. The aim of this study was to investigate the effect of this prolonged sitting on students’ self-perceived physical, mental, and cognitive condition and to answer the question of whether simple standing breaks in lectures can help students improve these conditions and for example feel more concentrated, motivated, or less tense in class. A five-minute standing break was introduced using a designed presentation slide for one semester in five different 90-min lectures. In addition, an active break as well as an open break with no trigger were implemented in two further lectures to explicitly investigate the effects of a standing break. Before, during, and after the semester, the students were surveyed about their physical, mental, and cognitive condition (836 respondents at start, 634 during semester, and 528 at the end). To evaluate the practicality and acceptance of the standing break, lecturers were interviewed about their experience. At all survey time points, the standing break was highly accepted by the university students. ... mehrAbout three quarters of the students felt a relaxation of the muscles in the neck and shoulder as well as in the back and the legs. More than three quarters perceived an increase in concentration, receptiveness and retentiveness, motivation, and well-being. Results of the statistical analysis indicate that a standing break as well as an active break are more effective than an open break to improve the self-perceived physical and psychological well-being of the university students. The increase in cognitive skills is reported by all groups, including the group who were offered open breaks. Hence, standing breaks in university lectures receive a high level of acceptance and practicability and have the potential to increase students’ physical, mental, and cognitive condition and contribute to students’ physical activity and health. While field research provides opportunities such as the testing of measures in the natural environment and producing real-life results relevant to the students and lecturers, it also imposes limitations as lecture settings differed, not all disturbances could be controlled, and the participation in the study might have led to social-desirability bias. For a sustainable development of a standing-friendly teaching and learning culture at universities, further interventions as well as the consideration of the topic in all processes and decisions within the universities are necessary. Since this study has taken place, student-life has changed drastically with COVID-19 measures. While this current paper is based on research conducted in 2019 and has only tested live lectures on campus, the tools tested could also be used for online lectures.