El Portalon de Cueva Mayor located in the UNESCO World Heritage Centre of Atapuerca, is a unique reference Late Prehistory archaeological site in the Iberian Peninsula, covering some 7 kyr of Holocene occupations. Herein we present the study of lipid residue analyses from 108 pottery sherds coupled with faunal kill-off patterns from the Neolithic, Chalcolithic and Bronze Age periods to gain new insights into natural product exploitation and animal management. The molecular and isotopic analyses of lipid extracts provide evidence for the use of carcass and dairy products at the site. The construction of kill-off profiles for the ovicaprids (sheep/goat) and bovines (cattle) provide complementary insights into animal management at the site, suggesting that both bovines and ovicaprids were exploited for their milk. The diachronic evolution of identified products show no major variations in livestock husbandry or diet due to cultural or environmental change. Changing uses and different activities at the cave over time (domestic, funerary, etc.) are likely responsible for the subtle differences identified in the results. This study provides the first insights into human subsistence strategies through time at El Portal ' on de Cueva Mayor.