Background: Cold atmospheric plasma (CAP) has proven its benefits in the reduction of various bacteria and fungi in both in vitro and in vivo studies. Moreover, CAP generated by dielectric barrier discharge (DBD) promoted wound healing in vivo. Charged particles, chemically reactive species (such as O3, OH, H2O2, O, NxOy), ultraviolet radiation (UV-A and UV-B), strong oscillating electric fields as well as weak electric currents are produced by DBD operated in air. However, wound healing is a complex process, depending on nutrient and oxygen supply via cutaneous blood circulation. Therefore, this study examined the effects of CAP on cutaneous microcirculation in a prospective cohort setting. Hypothesis: Cold atmospheric plasma application enhances cutaneous microcirculation. Methods: Microcirculatory data of 20 healthy subjects (11 males, 9 females; mean age 35.2 ± 13.8 years; BMI 24.3 ± 3.1 kg/m2) were recorded continuously at a defined skin area at the radial forearm. Under standardized conditions, microcirculatory measurements were performed using a combined laser Doppler and photospectrometry system. After baseline measurement, CAP was applied by a DBD plasma device for 90 s to the same defined skin area of 22.5 cm2. Immediately after the application cutaneous microcirculation was assessed for 30 min at the same site. Results: After CAP application, tissue oxygen saturation immediately increased by 24% (63.8 ± 13.8% from 51.4 ± 13.2% at baseline, p < 0.001) and stayed significantly elevated for 8 min. Cutaneous blood flow increased by 73% (41.0 ± 31.2 AU from 23.7 ± 20.8 AU at baseline, p < 0.001) and remained upregulated for 11 min. Furthermore, cutaneous blood flow showed two peaks at 14 (29.8 ± 25.0 AU, p = 0.049) and 19 min (29.8 ± 22.6 AU, p = 0.048) after treatment. Postcapillary venous filling pressure continuously increased, but showed no significant change vs. baseline in the non-specific BMI group. Subgroup analysis revealed that tissue oxygen saturation, postcapillary venous filling pressure and blood flow increased more in case of a lower BMI. Conclusion: CAP increases cutaneous tissue oxygen saturation and capillary blood flow at the radial forearm of healthy volunteers. These results support recently published data on wound healing after CAP treatment. However, further studies are needed to determine if this treatment can improve the reduced microcirculation in diabetic foot ulcers. Moreover, repetitive application protocols have to be compared with a single session treatment approach.