Background: Limiting T cell dysfunction is crucial for improving immunotherapy outcomes in patients with solid tumors, including non-small cell lung cancer (NSCLC). Hypoxia can occur in the tumor microenvironment (TME) and induce tumor infiltrating lymphocytes (TILs) adaptations, yet the impact of sustained hypoxia on human effector T cells remains poorly understood. Methods: Using Imaging Mass Cytometry (IMC), we simultaneously mapped 36 markers for cancer cells (DNA, H3, CK, Vimentin, PD-L1, PD-L2, FGL1, CD47, beta2M), immune cells (CD3, CD4, CD8, CD20, CD56, CD68, CD45RA, CD45RO, FOXP3, DC-Lamp), functional states (LAG-3, PD-1, TIM-3, VISTA, TBET, EOMES, TOX1/2, TCF7, CD25, CD27, CD137, GZB, ARG1, KI67 and CC3) and hypoxia (HIF1alpha, CA9) in 114 pre-treatment NSCLCs from two independent cohorts of patients treated with chemotherapy (Cohort 1, n=61) or PD-1 axis blockers (Cohort 2, n=53). Single-cell segmentation enabled the study of the relationship between hypoxia and effector T cell states. To assess functional changes, human PBMCs from healthy donors and in vitro expanded TILs obtained from primary human NSCLCs were exposed to recurrent TCR stimulation for 6 days under normoxia (21% O 2 ) or hypoxia (1% O 2 ). Longitudinal changes in effector T cell phenotype and function were evaluated by flow cytometry, scRNA/ATACseq, migration and cancer cell killing assays. Results: In primary NSCLCs, hypoxic CD8 + TILs showed heterogeneous distribution, higher activation and dysfunction markers (CD25, PD-1, LAG-3, TIM-3, TOX1/2, GZB, KI67), and distinct association with patient outcomes. Under normoxia, short-term (1 day) stimulation increased activation, cytokine production and cancer cell killing, followed by progressive acquisition of a dysfunctional state during intermediate (3 days) and long-term stimulation (6 days) characterized by increased markers of dysfunction, proliferation, reduced cytokine production, and impaired migration and cytotoxicity. Under hypoxia, short-term stimulation did not alter the T cell phenotype but reduced TNFalpha production, migration and tumor killing capacity compared to normoxia. However, prolonged T cell stimulation under hypoxia (3-6 days) led to a distinct phenotype with reduced dysfunction markers, increased cytokine production, and improved migration and killing. Differences were noted between PBMCs and TILs. Long-term stimulation under hypoxia showed distinct transcriptomic and epigenetic changes supporting specific mechanisms underlying the observed responses. Conclusions: Hypoxia modifies the activation and functional profile of effector TILs in NSCLC. Short and long-term hypoxia induces opposite phenotypic, functional and molecular changes on effector T cells during recurrent TCR stimulation. These results expand our understanding of the dynamic role of hypoxia on human T cell dysfunction with prominent biological and translational implications.