Background: Instrument-assisted manual therapy (IAMT) is indicated to improve flexibility, reduce pain, and induce hyperaemia locally and along myofascial chains. The underlying effects are largely unclear. This randomised, placebo-controlled pilot study aimed to gain first insights into these effects, primarily on the structural level, through ultrasonography. Methods: 67 healthy female soccer players aged 20.9 (±3.9) years were examined after right lumbar intervention (IAMT: intervention group (IG), heat application: comparison group (CG), pressure-less placebo: placebo group (PG)). Ultrasonography (absolute movement and shear motion), flexibility tests (passive straight leg raise test (PSLR), lumbar and thoracic double inclinometry), and superficial skin temperature were recorded before (t0), immediately (t1) and 45 min after the intervention (t2). Results: IAMT decreased the absolute mobility of the superficial lamina and its shear motion to the superficial fascia compared with the PG (t1; p < 0.05). PSLR improved in the IG compared with the CG (t2) and PG (t1, t2; p < 0.05). The temperature increased in the IG and CG compared with the PG (t1, t2) and in the CG compared with the IG (t1; p < 0.05). Conclusion: IAMT of the lumbar back briefly reduces absolute mobility of the superficial lamina and its shear motion to the superficial fascia, improves flexibility, and increases the temperature.