Preventing dehydration during in vitro testing of isolated layers of annulus fibrosus tissue may require different test conditions than functional spine units. The purpose of the study was twofold: (A) to quantify changes in mass and thickness of multilayer annulus samples in four hydration environments over 120 min; and (B) to quantify cycle-varying biaxial tensile properties of annulus samples in the four environments. The environments included a saline bath, air, relative humidity control, and misting combined with controlled humidity. The loading protocol implemented 24 cycles of biaxial tensile loading to 20% strain at a rate of 2%/s with 3-, 8-, and 13-min of intermittent rest. Specimen mass increased an average (standard deviation) 72% (11) when immersed for 120 min (p < 0.0001). The air condition and the combined mist and relative humidity conditions reduced mass by 45% (15) and 25% (23), respectively, after 120 min (p < 0.0014). Stress at 16% stretch in the air condition was higher at cycle 18 (18 min of exposure) and cycle 24 (33 min of exposure) compared to all other environments in both the axial and circumferential directions (p < 0.0460). There was no significant change in mass or thickness over time in the relative humidity condition and the change in circumferential stress at 16% stretch between cycles 6 and 24 was a maximum of 0.099 MPa and not statistically significant. Implementation of a controlled relative humidity environment is recommended to maintain hydration of isolated annulus layers during cyclic tensile testing.