© 2022 Elsevier LtdIntroduction: Cold spray is a form of cryotherapy used in acute injuries at sports medicine. In the current study, we aimed to investigate the acute effect of cold spray application as a treatment strategy for acute sports injuries on the mechanical properties of the rectus femoris muscle in athletes. Methods: The study included 23 volunteer active male athletes between the ages of 18–23 with a subepidermal fold thickness of the rectus femoris muscle between 5 mm and 15 mm. Skin temperature (thermal camera) and mechanical property evaluations of the muscle (Myoton Pro) were measured before, immediately after, and in 2 min, 5 min, 10 min and 15 min intervals following cold spray application. Results: The skin temperature value of the athletes were significantly lower even in the 15-min interval following application (p < 0.001). Muscle tone increased significantly after the application and in the second minute compared to the pre-application (p < 0.001). Muscle stiffness increased significantly only after the second and fifth minutes compared to the pre-application (p < 0.001). Muscle decrement showed an increase in all time-points compared to the pre-application (p < 0.05). Conclusion: In athletes, the rectus femoris muscle has been observed to become harder and less elastic following cooling with cold spray. These changes did not improve completely following the 5 min. The muscle regained its mechanical properties at the earliest approximately 10 min after the cold spray application.