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The aim of this work is to investigate the changes in the physicochemical properties of hydroxyapatite (HAp) extracted from horse humerus bones of different ages (1, 3, 6, and 8 years) subjected to low temperature calcination (600°C). Thermal analysis revealed significant mass loss due to water, collagen, organic compounds, carbonates, and age-related magnesium out-diffusion. Higher fat content in older bones contributed to increased mass loss. Phosphorus content remained constant across age groups, while calcium and sodium showed age-related fluctuations. Magnesium levels decreased with age, emphasizing its importance for early bone development. The Ca/P ratio deviated from the stoichiometric values due to additional ions from biogenic sources. Infrared spectroscopy identified functional groups in carbonated HAp, with changes observed before and after calcination. The full width at half maximum (FWHM) of the 961 cm -1 band decreased with age, indicating improved crystalline quality. The molar absorption coefficients provided information on the changes in molecular concentration and emphasized the differences between the age groups. X-ray analysis revealed nanocrystalline HAp in all samples, with crystallite size increasing with age. Rietveld analysis showed that the lattice parameters were affected by the presence of organic material, but the lattice constants remained stable, confirming high crystallinity independent of age. TEM analysis confirmed nanocrystalline structures, with crystallite size increasing with age. SEM images showed the characteristic porosity of calcined HAp, with particle size correlating positively with age. Calcination at 600°C preserved the nanoscale properties and microcrystal formation. Raman spectroscopy confirmed the identity of HAp, with FWHM variations indicating age-related changes in crystalline quality. EHAp1 showed increased FWHM, indicating lower crystalline quality and increased trace element content., (© 2024 The Author(s). Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)