This study was designed to evaluate changes in erythrocyte contents during endurance moderate intensity exercise, a model of physiological oxidative stress. 16 endurance-trained subjects cycled 2 h at 55% of maximal aerobic capacity and blood was collected every 15 min. Transmission FT-IR spectrometry was used to analyze separately plasma and erythrocyte content changes during oxidative stress. Erythrocyte FT-IR spectra were corrected for hemoconcentration (Hc) before spectral areas integration of main IR absorbances belonging to phospholipids [nu(as)(CH(3)), [nu(as)(CH(2)), and nu(P=O)], proteins [nu(C=O) and delta(N-H)], and lactate [nu(C-O)] were used to determine erythrocyte content changes. Changes in nu(as)(CH(2)) and nu(P=O) absorbances while nu(as)(CH(3)) remained stable showed the magnitude of free radical attacks on phospholipids bilayer. Decrease in nu(C=O) and delta(N-H) absorbances while plasma and intracellular lactate, O(2) consumption, and Hc rose were linked to hemoglobin, and possibly spectrin, denaturation. Finally, the synergistic changes found between physiological, plasmatic and erythrocyte parameters showed that FT-IR spectrometry was a sufficiently accurate and sensitive method to determine acute changes in erythrocytes during moderate, physiological, oxidative stress.