The objective of this study was to assess whether different lead placement patterns and frequencies (measured in hertz, Hz) in electro-acupuncture (EAP) significantly influence tissue vascular perfusion. Thirty-six clinically healthy dogs from a local dog sport community were enrolled in the study. Each subject received 6 EAP treatments, and a 7th treatment which was control (no needle placement). A total of 10 needles were placed at Back-shu points (BL-20 through BL-24) bilaterally for 20 minutes. The 6 EAP techniques included lead placement that was contralateral (BL-20 left side to BL-20 right side) or ipsilateral (BL-20 left side to BL-24 left side) and frequency settings at 20, 120, and 80-120 Hz. For each subject, the treatment order was randomized with a 2-week washout period between treatments to avoid carry-over effect. Pre- and postacupuncture skin temperature changes, measured by infrared thermography, were compared among the 7 treatments. All EAP treatments resulted in significant skin temperature increase (p<0.05). No significant temperature change resulted from the no-treatment control (p=0.983). The “EAP 20 Hz contralateral” stimulation had significantly greater temperature increase than the other acupuncture treatment methods, with greatest to lowest temperature increase as follows: 20 Hz contralateral, 80-120 Hz ipsilateral, 20 Hz ipsilateral, 80-120 Hz contralateral, 120 Hz contralateral, 120 Hz ipsilateral. Study results indicate that both pattern of lead placement and the frequency used in EAP affect tissue perfusion. These findings are unique and could help shape best practices for selecting EAP frequency and lead placement in future applications.