Background: The myofascial release technique is known to be an effective technique for increasing posterior fascia flexibility in short hamstring syndrome (SHS) subjects. But therapeutic mechanism of myofascial relaxation remains unclear. Recently, the theory of autonomic nervous system domination has been raised, however, a proper study to test the theory has not been conducted. Objectives: To investigate whether the application of the myofascial release technique can induce changes in the autonomic nervous system and affect the secretion of stress hormones and myofascial relaxation. Design: Quasi-experimental study. Methods: Twenty-four subjects with SHS were randomly divided into two groups. In the experimental group, the suboccipital muscle inhibition (SMI) technique was applied to the subjects for 4 min in supine position, and in the control group, the subjects were lying in the supine position only. A forward flexion distance (FFD) was conducted, blood pressure, heart rate, and cortisol levels were measured before and after the intervention and 30 min after intervention to determine myofascial relaxation and stress hormone levels. The evaluation was conducted separately in blind by an evaluator. Results: A FFD decreased in the experimental group, no change in cortisol was observed. On the contrary, a decrease in cortisol appeared in the control group after 30 minutes. Conclusion: The myofascial release technique is an effective treatment to increase the range of motion through posterior superior myofascial chain, but there is no evidence that myofascial release technique can control the autonomic nervous system.