SignificanceLaser ablation MC-ICP-MS allows in situ strontium isotope data to be obtained for incrementally formed bioapatites such as enamel with extremely high spatial resolution. Here, we provide a large-scale application of the method comparing the mobility and subsistence behavior of Middle and Upper Paleolithic humans in the same landscape. These remains and the fauna analyzed alongside come from the Almonda karst system (Portuguese Estremadura). Data suggest that regional Middle Paleolithic individuals roamed across a subsistence territory of approximately 600 km2, while Upper Paleolithic individuals moved seasonally and exploited a smaller territory of approximately 300 km2.