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The primary method previously used by paleontologists to estimate climatic changes that occurred during Pleistocene glacial cycles was the determination of ¹⁸O/¹⁶O ratios in calcareous fossils. However, because this ratio is influenced by a number of factors, the absolute magnitude of the temperature difference between Pleistocene glacial and interglacial cycles could not be unequivocally ascertained. For example, both temperature fluctuations and isotopic changes in seawater affect the ¹⁸O/¹⁶O ratio. And, since both factors influence the ratio in the same direction, the contribution of each to the ¹⁸O/¹⁶O cannot be determined.

Fortunately, recent studies indicate that the racemization reaction of amino acids can be used to determine more accurately temperatures that occurred during Pleistocene glacial cycles. Only L-amino acids are usually found in the proteins of living organisms, but over long periods of geological time these acids undergo racemization, producing D-amino acids, which are not found in proteins. This reaction depends on both time and temperature; thus, if one variable is known, the reaction can be used to calculate the other.