Background: Core muscles provide important dynamic stabilization of the lumbar spine. The twist sit-up can simultaneously stimulate the external oblique (EO), internal oblique (IO), and transverse abdominis (TrA) muscles. Objectives: The purpose of this study is to investigate the impact of knee angles during twist sit-up exercises and determine the most efficient knee angle for performing this exercise. Design: Cross-sectional study. Methods: The study recruited 30 students from S University regardless of gender. The subjects performed a total of 10 twist sit-ups at five different knee angles (0, 40, 65, 90, and 105 degrees), divided between the ipsilateral and contralateral sides. The thickness of abdominal muscles (EO, IO, and TrA), was measured using ultrasonography on rest position and full contraction position for each angle of knee flexion and compared between angles. Results: There were significant differences in the ipsilateral EO, IO, and TrA muscle thickness at different knee flexion angles (P<0.5). There was a significant difference in the thickness of EO between 0 and 105 degrees and between 40 and 105 degrees (P<0.5), a significant difference in the thickness of IO between 0 and 105 degrees (P<0.5), and a significant difference in the thickness of TrA between 0 and 105 degrees, 40 and 90 degrees, and 65 and 90 degrees (P<0.5). Conclusion: In conclusion, the muscles involved in axial rotation i.e., EO and IO, maximally contract at a knee flexion angle of 105 degrees. Therefore, this may be the most optimal angle to perform twist sit-ups.