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Background The most widely used graft as a replacement in anterior cruciate ligament (ACL) reconstruction is the semitendinosus (ST) tendon graft. After harvesting for ACL reconstruction, the hamstring tendon regenerates in most people and becomes similar to normal. The effect of graft harvest on muscle morphology and function remains unclear. The present study aimed to examine the morphology of the ST during isometric contraction after harvesting the ST tendon for ACL reconstruction. Methods Maximal isometric contractions of the knee flexors from two angular positions were performed by 8 participants, at least 1 year after ACL reconstruction with an ST tendon graft and 8 matched controls. Ultrasonographic images were used to measure the pennation angle and muscle thickness of the ST muscle. Results There was not a statistically significant difference in pennation angle values between the control group and the group who underwent ACL reconstruction (p >0.05). Muscle thickness was significantly higher in the ACLR group compared with controls (p Conclusions Individuals who underwent ACL reconstruction display a higher ST muscle thickness but similar pennation angle compared with controls. This indicates that ACL reconstruction has an effect on ST muscle belly but effect on force generation capacity is rather limited. Level of evidence IIb.