Objective: The purpose of the present study was to compare the effects of traditional and intermittent physical fitness-based teaching units on secondary school students’ motivation toward Physical Education, autotelic experience, and physical self-concept. Methods: Six classes [126 students (57.9% females) aged 13-15 years], balanced by grade, were cluster-randomly assigned into control (n = 40), traditional (n = 34) and innovative (n = 52) groups. The traditional group performed a physical fitness teaching unit twice a week for nine weeks (35-40 minutes of the main part of each session). The innovative group worked during the first half of the sessions’ main part (18-20 minutes) similarly to the traditional group, and during the second half they worked on invasion sports. Before and after the intervention, students’ motivation toward Physical Education and physical self-concept were measured through the Perceived Locus of Causality-II Scale and the short form of the Physical Self Description Questionnaire. Results: The Multilevel Linear Model showed that the control group students decreased statistically significantly in the integrated, introjected, and controlled motivation compared to those from the innovative and traditional groups (p < 0.05; d = 0.17-0.51). However, no statistically significant differences in the other motivational dimensions, nor on the autotelic experience and physical self-concept dimensions were found between the three groups (p > 0.05). Conclusion: Regardless of the teaching unit structure applied (i.e., traditional or intermittent), it could be necessary that Physical Education teachers apply specific strategies for improving these psychological variables of the students.