Rationale: Chemical pre-treatment is an important methodological step aimed to remove exogenous materials introduced to archaeological tooth enamel through various diagenetic pathways prior to carbon and oxygen isotope analysis. Concerningly, some of these pre-treatment methods (NaClO, H ₂O ₂ and CH ₃COOH) have been shown to cause significant changes to enamel chemical properties and stable isotope values. This study aims to re-examine the effects of commonly used pre-treatment protocols applied to the enamel structure on bioapatite δ ¹³C and δ ¹⁸O values, as well as investigate patterns related to site burial context and sample preservation, as indicated by pre-screening using Fourier Transform Infrared (FTIR) spectroscopy. Methods: Modern and archaeological samples were subjected to ten commonly used pre-treatment protocols using NaClO, H ₂O ₂ and CH ₃COOH at various time intervals. Preservation status and diagenetic alteration prior to and after treatment were investigated using Attenuated Total Reflectance- Fourier Transform Infrared Spectroscopy (ATR-FTIR). δ ¹³C and δ ¹⁸O values were measured before and after the treatment to determine if treatments induced isotopic shifts. Results: The results show that all pre-treatment protocols caused shifts in δ ¹³C and δ ¹⁸O values in the range of 0‰ to ±1.5‰ in both archaeological and modern samples. Most treated samples also shown increased crystallinity, likely indicating sample recrystallisation. We argue that these changes are indicative of more than just removal of contamination and diagenetic alterations, but also of dissolution and/or restructuring of the enamel carbonate leading to changes to the in vivo isotope signal. Conclusions: We recommend careful consideration of sample burial context and possible routes of diagenesis when choosing a pre-treatment protocol. Use of H ₂O ₂ and NaClO to remove organic matter from samples is discouraged as it incurs unwanted changes to the enamel chemical properties and isotope values. We also recommend use of only short acetic acid treatment protocols to avoid possible recrystallisation during prolonged contact.