This study aimed to compare the effects of myofascial release (MFR) and hot pack therapy (HPT) on fascial gliding and flexibility of the vastus lateralis muscle. Methods: Three treatments were applied to the left vastus lateralis muscles of each participant (12 healthy males): MFR for 4min, superficial HPT for 10min, and superficial HPT for 20min. Deep fascial motion was measured by B-mode ultrasound, whereas muscle stiffness was measured by real-time elastography (RTE) and a durometer before and after the interventions. Results: Only MFR resulted in changes in both deep fascial motion and muscle stiffness measured by RTE. Durometer-measured muscle stiffness revealed changes following both MFR and 20-min HPT but not 10-min HPT. Conclusions: HPT may produce only superficial effects. Because MFR improved all measured parameters, continuous stretching and pressure are probably important for improving fascial gliding and flexibility of the vastus lateralis muscle.