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Cold atmospheric plasma (CAP) reduces bacteria and interacts with tissues and cells, thus improving wound healing. The CAP-related induction of neutrophils was recently described in stained sections of wound tissue in mice. Consequently, this study aimed to examine the functionality of human polymorphonuclear cells (PMN)/granulocytes through either a plasma-treated solution (PTS) or the direct CAP treatment with different plasma modes and treatment durations. PTS analysis yielded mode-dependent differences in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) after CAP treatment. Live-cell imaging did not show any chemo-attractive or NETosis-inducing effect on PMNs treated with PTS. The time to maximum ROS production (T_maxROS) in PMNs was reduced by PTS and direct CAP treatment. PMNs directly treated with CAP showed an altered cell migration dependent on the treatment duration as well as decreased T_maxROS without inducing apoptosis. Additionally, flow cytometry showed enhanced integrin and selectin expression, as a marker of activation, on PMN surfaces. In conclusion, the modification of PMN immunoreactivity may be a main supporting mechanism for CAP-induced improvement in wound healing.