Although the clinical use of electro-acupuncture is widespread, the neuromodulating influence of various applied frequencies is not well established. Heart rate variability (HRV), stress and parasympathetic recovery are closely related to health, longevity and vitality in humans. This study was undertaken to determine the influence of different electro-therapy frequencies on various autonomic nervous system markers when applied to a Battlefield Acupuncture Protocol (BFA). A detailed comparison of autonomic nervous system (ANS) response to low frequency (LF) 2.5 Hz electro-acupuncture and mid-frequency (MF) 15 Hz electro-acupuncture applied by point stimulation to acupuncture points was undertaken on 2 groups of 12 patients. Both LFEA and MFEA were applied to Battlefield Acupuncture protocol, consisting of five (5) key acupuncture ear (auricular) points that isolate the autonomic nervous system (ANS) and central nervous system's role in the chronic/acute pain cycle. Evaluations entailed an advanced status of autonomic nervous system (ANS) function through Electro-Cardiogram (ECG) baseline markers reflecting: sympathetic stress (SI); parasympathetic vagal tone (high frequency [HF]) and heart rate variability (HRV = total power). All were repeated subsequent to electro-therapy using 2 separate electro-modalities of low-frequency (2.5 Hz) electro-acupuncture (LFEA) and Mid-Frequency (15 Hz) electro-acupuncture (MFEA). All 24 patients received one (1) elector-acupuncture session. The autonomic nervous system response to LFEA (2.5 Hz) reflected a statistically significant pre-post improvement in three of the markers collected: heart rate variability (HRV) improved by 61% [ = 0.002]; sympathetic stress (SI) reduced 42% [ = 0.002]; and parasympathetic vagal tone (HF) increased 56% [ = 0.017]. In contrast, MFEA (15 Hz) showed positive but non-significant changes in outcomes in all nervous system markers. The autonomic nervous system response with LFEA showed a measurable reduction in sympathetic stress with subsequent improvement in vagal tone, and HRV. This positive sympathetic nervous system deactivation from LFEA application shown in this study could have a major impact on other pathologies related to human health and longevity. Further cohort studies are warranted to determine the validity of these outcomes.