INTRODUCTION: Negative pressure wound therapy (NPWT) is utilized early after soft tissue injury to promote tissue granulation and wound contraction. Early post-injury transfers via aeromedical evacuation (AE) to definitive care centers may actually induce wound bacterial proliferation. However, the effectiveness of NPWT or instillation NPWT in limiting bacterial proliferation during post-injury AE has not been studied. We hypothesized that instillation NPWT during simulated AE would decrease bacterial colonization within simple and complex soft tissue wounds. METHODS: The porcine models were anesthetized before any experiments. For the simple tissue wound model, two 4-cm dorsal wounds were created in 34.9 ± 0.6 kg pigs and were inoculated with Acinetobacter baumannii (AB) or Staphylococcus aureus 24 hours before a 4-hour simulated AE or ground control. During AE, animals were randomized to one of the five groups: wet-to-dry (WTD) dressing, NPWT, instillation NPWT with normal saline (NS-NPWT), instillation NPWT with Normosol-R® (NM-NPWT), and RX-4-NPWT with the RX-4 system. For the complex musculoskeletal wound, hind-limb wounds in the skin, subcutaneous tissue, peroneus tertius muscle, and tibia were created and inoculated with AB 24 hours before simulated AE with WTD or RX-4-NPWT dressings. Blood samples were collected at baseline, pre-flight, and 72 hours post-flight for inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor alpha. Wound biopsies were obtained at 24 hours and 72 hours post-flight, and the bacteria were quantified. Vital signs were measured continuously during simulated AE and at each wound reassessment. RESULTS: No significant differences in hemodynamics or serum cytokines were noted between ground or simulated flight groups or over time in either wound model. Simulated AE alone did not affect bacterial proliferation compared to ground controls. The simple tissue wound arm demonstrated a significant decrease in Staphylococcus aureus and AB colony-forming units at 72 hours after simulated AE using RX-4-NPWT. NS-NPWT during AE more effectively prevented bacterial proliferation than the WTD dressing. There was no difference in colony-forming units among the various treatment groups at the ground level. CONCLUSION: The hypoxic, hypobaric environment of AE did not independently affect the bacterial growth after simple tissue wound or complex musculoskeletal wound. RX-4-NPWT provided the most effective bacterial reduction following simulated AE, followed by NS-NPWT. Future research will be necessary to determine ideal instillation fluids, negative pressure settings, and dressing change frequency before and during AE.