•Myofascial trigger points (MTrPs) are hyperirritable spots in skeletal muscle, commonly in hypermobile people.•Musculoskeletal interfiber counterirritant stimulation (MICS) is used through ischemic compression.•Autonomic nervous system (ANS) activity caused an increase in vagal tone and a decrease in sympathetic tone.•MICS technique reduces the pain perception intensity in MTrPs modulates the ANS activity, and increase heart rate variability (HRV). Joint hypermobility (JH) conditions suggest dysfunction in the autonomic nervous system (ANS) (dysautonomia), associated with multifactor non-articular local musculoskeletal pain, and remains a complex treatment. This study aims to determine the effects of musculoskeletal interfiber counterirritant stimulation (MICS) as an innovative treatment of myofascial trigger points (MTrPs) on the upper trapezius muscle in JH patients. We evaluate the ANS activity by wavelet transform spectral analysis of heart rate variability (HRV) in sixty women, equally divided: MTrP, MTrP + general joint hypermobility (GJH), and MTrP + joint hypermobility syndrome (JHS). The protocol phases were rest, stimulation, and recovery, with clinical and home treatment for three-days. All groups show a significantly decreased in pain perception during and post-treatment, and an increased parasympathetic ANS activity under MICS in the GJH and JHS groups. The variables low-frequency (LF) vs. high-frequency (HF) showed significant differences during the protocol phases, and the LF/HF ratio maintained a predominance of sympathetic activity (SA) throughout the protocol. The new MICS technique reduces the pain perception and modulates the ANS activity by an increase in vagal tone, and a decrease in sympathetic tone. This modulation was followed by an increase in the HRV in JH patients after treatment with MICS. Clinical Trials: RBR-88z25c5.