Introduction: Instrument-assisted manual therapy (IAMT) is suitable for optimizing movement prerequisites, such as range of motion, flexibility, microcirculation, and pain inhibition along myofascial chains, potentially leading to a reduction in hamstring injuries. To date, however, IAMT's modes of action remain largely unclear. This study aimed to examine the functional and structural effects of repetitive IAMT after 2.5 and five weeks. Methods: Sixteen healthy female soccer players (age: 21.4 (±5.1) years) were randomly grouped into an intervention group and a placebo control group. The intervention group received nine IAMT sessions twice weekly at the right lumbar region. The placebo group received a single pressureless "therapy" at baseline. In addition to structural ultrasound analyses (absolute motion and shear motion), functional mobility tests (passive straight leg raise (PSLR) test and lumbar and thoracic double inclinometry) were performed 4.0 (±2.0) days after the fourth IAMT and 3.4 (±1.1) days after the ninth IAMT in both groups. Results: Hamstring flexibility in the PSLR test improved significantly after the ninth IAMT compared with the placebo group (p < 0.05, effect size: 0.913). No systematic differences were seen at the structural level. Conclusion: Repetitive IAMT can improve hamstring flexibility. Further studies in larger groups and diverse collectives are necessary to additionally test the postulated preventive effect also on hamstring injuries. Whether ultrasound is the right method for detecting structural changes in this context needs to be verified in the future.