PB1-F2 is a non-structural protein of influenza A viruses (IAV) that modulates viral pathogenesis in a host-specific manner. In mammals, this protein has been shown to increase IAV virulence by delaying the early immune response and, eventually, exacerbating lung inflammation at the late stage of infection. PB1-F2 is a small protein, but displays very high sequence polymorphism and sequence length disparity depending on viral strain. These features result in strong variations in the cellular activity of PB1-F2. Studies have also reported that the effect of PB1-F2 is cell-type dependent. It has notably been shown that PB1-F2 can promote apoptosis in immune cells, but not in epithelial cells. This phenomenon appears to be partly related to the higher order structure of the protein, given that the presence of PB1-F2 β-aggregated structures in infected immune cells correlates with cell death induction. In this work, we evaluated, by synchrotron Fourier transform infrared spectromicroscopy, the impact of the transient expression of PB1-F2 on the biochemical composition of the human epithelial cell line HEK293T. Two PB1-F2 variants that are closely related to each other but derived from a strain with high [A/BrevigMission/1/1918 (H1N1)] or a low [A/WSN/1933 (H1N1)] virulence were studied here. Infrared spectra analysis revealed no specific enrichment of β-aggregated structures in PB1-F2-expressing cells. Nevertheless, this analysis suggested that there is a higher content of β-sheet secondary structures in the PB1-F2 from A/WSN/1933 than that from A/BrevigMission/1/1918. Our data also showed no change in membrane composition in the presence of PB1-F2, implying that PB1-F2 does not promote apoptosis in HEK293T cells. Finally, we found that the PB1-F2 from A/WSN/1933 interferes with adenosine triphosphate production, suggesting that this PB1-F2 variant may disturb the mitochondrial activity.