Bicontinuous microemulsions (BMEs), self-assembly systems consisting of oil and water nanodomains separated by surfactant monolayers, have many applications. However, changes in structure and properties of BMEs in the vertical direction can affect BMEs' utility. This study's objective was to determine the effect of equilibration time (τeq) on structural changes in the vertical direction for bicontinuous phases of Winsor-III (WIII) systems in situ or after being isolated, for D2O + H2O/1-dodecane/sodium dodecyl sulfate (SDS)/1-pentanol/NaCl at 22 °C. Small-angle neutron scattering (SANS) measurements were performed using a vertical stage sample environment that precisely aligned samples in the neutron beam. SANS data were fitted by the Teubner-Strey (TS) model and changes in TS-derived parameter values were observed. For 10 min ≤ τeq ≤ 4 h, the effective activity of the bicontinuous phase's surfactant monolayers increased with time at all vertical positions. At short equilibration (τeq = 10 min), small but significant amounts of water and oil were transiently emulsified near the WIII upper liquid-liquid interface. WIII systems underwent a relaxation process after being transferred to narrow 1 mm pathlength cells, resulting in a decrease of surfactant activity for the top half of the bicontinuous phase. For isolated bicontinuous phases, results suggest that SDS was desorbed from the BMEs by quartz near the bottom, while near the top, the water concentration near was relatively high. The results suggest that WIII systems should equilibrate for at least 4 hours after being prepared and transferred to a container that differs in cross sectional area and surfactant behavior in BMEs can change near interfaces.