Fourier transform infrared (FTIR) microspectroscopy is assessed in terms of two techniques (i.e., transmission and transflection) as a method for rapid measurements of blood plasma. Apart from the expected effect of the electric field standing wave (EFSW), we also noticed that second-derivative IR spectra recorded in transflection mode exhibited a significant shift in the amide I band (up to 1667 cm−1) in comparison to the one recorded in transmission (1658 cm−1). This has not been reported thus far in studies of the EFSW distortion of IR spectra of biological material. The thinner the sample deposited on the low-e microscope slide, the lower the position of the amide I band found in FTIR spectra, suggesting various plasma compositions after stratification or certain changes in secondary protein conformations due to chemical and/or physical effects. There are potentially several phenomena that can occur at the surface of both IR substrates affecting the protein profile, including changes in optical properties (refractive index), variation in water content in the sample, and segregation of plasma components. All three hypotheses are discussed here, with the help of atomic force microscopy (AFM).