Background: Immune normalization has emerged as a new paradigm in immunotherapy, which is proposed in cancer patients instead of the traditional immune enhancement therapy. The immune normalization strategy may also be implemented in cancer prevention of sub-healthy individuals.Methods: We established in vitro cultured mixed-Natural Killer cells (NKM), which could be used to achieve immune normalization. We defined the sub-healthy individuals after analyzing the PD-1 ratio in PBMCs from 95 donors over 50 years of age.Results: NKM were composed by approximately 20% NK cells (CD3-CD16+ or CD56+), 30% NKT-like cells (CD3+CD16+ or CD56+) and other T cells. The in vitro cytotoxicity of NKM was ten times higher than the peripheral blood mononuclear cells (PBMC). NKM cytotoxicity was negatively correlated with the ratio of regulatory T cells (CD3+CD4+ T), and positively correlated with the ratio of NK cells, especially CD56brightCD16bright NK cells. We found the sub-healthy individuals displayed significantly higher ratio of CD3+PD-1+ T cells in PBMC (Ratio > 4%) and higher ratio of CD3+CD8+PD-1+ T cells in CD3+CD8+ T cells (Ratio > 10%) than the healthy controls. Then, we evaluated the potential clinical application of NKM therapy in one pancreatic cancer patient and four sub-healthy individuals.Conclusions: NKM therapy showed good tolerance and no side effects were found. In sub-healthy individuals, the ratio of CD3+PD-1+ T cells and CD3+CD8+PD-1+ T cells was significantly reduced after NKM treatment, which indicated that NKM therapy could potentially be used for cancer prevention and health care, thereby achieving the immune normalization.Trial registration: International Research Center for Regenerative Medicine, BIH-2018-1001. 01 August 2018, BOAO International Hospital.International Research Center for Regenerative Medicine, BIH-2018-1002. 01 August 2018, BOAO International Hospital.