In the current study, using self-determination theory as a theoretical framework, we used multiple real-time assessments to investigate the dichotomous relationship between autonomy need satisfaction and frustration, and how these constituents underpin student functioning among higher education students during a learning activity. In total, 124 STEM students were recruited from mandatory calculus seminars. The session lasted 90 minutes, with three fixed time measurements. Results indicate that satisfaction of the need for autonomy positively predicted vitality, situational interest, and perceived value, whereas autonomy need frustration was negatively related to the same outcome variables. Further, our results indicate fluctuations in both autonomy need satisfaction and need frustration during the calculus seminar. The results highlight well-differentiated effects of satisfaction and frustration of autonomy need and show that perceived need satisfaction and frustration constitute principal components in predicting student functioning.