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Violet laser acupuncture was recently described by our research group. Neurochemical studies of acupuncture analgesia revealed that 2- and 100-Hz peripheral stimulation induce differential peptide release from preproenkephalin and preprodynorphin via different neural pathways. The goal of the fifth part of this series was to perform a pilot investigation on the effects of 2- and 100-Hz violet laser acupuncture on heart rate (HR) and its variability (HRV) in 13 healthy volunteers (mean age ± standard deviation: 23.9 ± 1.7 years) using a new non-invasive method. We found a significant difference in mean HR before, during, and after 2- or 100-Hz violet laser acupuncture, and there was a significant difference of the low frequency/ high frequency ratio before, during, and after 2- or 100-Hz violet laser acupuncture. However, we did not observe a significant difference in total HRV before, during, or after treatment. Further studies should be carried out to compare the effects of different violet laser acupuncture frequencies.