Heat stress in hyper-prolific lactating sows is recognised as a factor reducing feed intake, milk production, and welfare, with significant losses in farm productivity. Individual capacities for body thermoregulation during environmental hyperthermia determine the adaptation of the animal during long and recurrent events. This study aimed to evaluate the ability of attenuated total reflectance (ATR) mid infrared (MIR) spectroscopy as a high-throughput method to identify markers of stress in plasma and milk collected from lactating sows under heat stress conditions fed with two levels of protein in the diet defined as low (16%) and standard (20%). The MIR spectra were analysed using linear discriminant analysis (LDA) and principal component analysis and validated using cross-validation. The results obtained indicated that MIR spectroscopy, in combination with chemometrics, was able to identify changes in the spectra associated with heat stress in wavenumbers corresponding with amide groups (proteins) (highest loadings observed in the regions between1065 and 1635cm-1), lipids and unsaturated fatty acids (regions between 1746 and 3063cm-1), lipo-polysaccharides (in 1247cm-1) and carbohydrates (around the region1050 cm-1). These results also indicated that the information provided by these wavenumbers can be used as metabolic markers of the adaptation of the sows to hyperthermia. It was concluded that MIR spectroscopy is a rapid and inexpensive tool capable of detecting and evaluating the main biochemical changes of hyperthermia on lactating sows, facilitating the development of palliative management strategies such as dietary manipulations.