Female ageing leads to reduced hair density and thinner fibres. The impact of the ageing dermal environment on the hair follicles (HFs) remains unclear. This study documents in situ changes in human female scalp skin of women (19-81 years (yrs)), and corresponding primary cultures of dermal fibroblast (DF) and dermal sheath (DS) cells. In situ, the papillary/reticular boundary was indistinguishable in young scalp, but delineated over 40yrs, with reduced rete ridges, changes in collagen organisation, reduced podoplanin (PDPN) and increased versican (VCAN) expression. Hyaluronic acid synthase 2 (HAS2) was highly expressed throughout the scalp. Matrix Metallopeptidase 1 (MMP1) and Metallopeptidase inhibitor 1 (TIMP1), cyclin-dependent kinase inhibitor 2A (p16INK4A), 11β-Hydroxysteroid dehydrogenase type 1 and 2 (11β-HSD1/HSD11B1 and 11β-HSD2/HSD11B2) mRNA expression increased in aged DFs. In DS cells, HAS2, Vimentin (VIM) ,PDPN, Lysosomal-associated membrane protein 1 (LAMP1), Sequestosome- 1 (P62) and Protease nexin-1 (SERPINE2) increased, while α-smooth muscle actin (aSMA) decreased. Both cell types exhibited elevated cartilage oligomeric protein (COMP) mRNA expression. Proteomics revealed elevated COMP expression in the DF secretome with age, suggesting a more fibrotic phenotype or DS migration into the dermis. DF and DS lysate protein expression suggests altered extracellular matrix (ECM) remodelling due to increased levels of MMP-2 and Protease inhibitor Plasminogen activator inhibitor-1 (Serpin E1/PAI-1). Cathepsin C/DPPI protein lysate expression decreased in DFs but increased in DS. In summary, ageing female scalp shows striking structural and biological changes in the HF environment that may impact hair growth, due to alterations in ECM, senescence and autophagy associated biomarkers.