Although deep roots can contribute substantially to whole-tree water use, little is known about deep root functioning because of limited access for in situ measurements. We used a cave system on the Edwards Plateau of central Texas to investigate the physiology of water transport in roots at 18–20 m depth for two common tree species, Quercus fusiformis and Bumelia lanuginosa. Using sap flow and water potential measurements on deep roots, we found that calculated root hydraulic conductivity (RHC) fluctuated diurnally for both species and decreased under shading for B. lanuginosa. To assess whether these dynamic changes in RHC were regulated during initial water absorption by fine roots, we used an ultra-low flowmeter and hydroxyl radical inhibition to measure in situ fine root hydraulic conductivity (FRHC) and aquaporin contribution to FRHC (AQPC), respectively. During the summer, FRHC and AQPC were found to cycle diurnally in both species, with peaks corresponding to the period of highest transpirational demand at midday. During whole-tree shade treatments, B. lanuginosa FRHC ceased diurnal cycling and decreased by 75 and 35% at midday and midnight, respectively, while AQPC decreased by 41 and 30% during both time periods. A controlled growth-chamber study using hydroponically grown saplings confirmed daily cycling and shade-induced reductions in FRHC and AQPC. Winter measurements showed that the evergreen Q. fusiformis maintained high FRHC and AQPC throughout the year, while the deciduous B. lanuginosa ceased diurnal cycling and exhibited its lowest annual values for both parameters in winter. Adjustments in FRHC and AQPC to changing canopy water demands may help the trees maintain the use of reliable water resources from depth and contribute to the success of these species in this semi-arid environment.