Purpose: To demonstrate how reference data affect the quantification of the apparent diffusion coefficient (ADC) in long diffusion time measurements with diffusion-weighted stimulated echo acquisition mode (DW-STEAM) measurements, and to present a modification to avoid contribution from crusher gradients in DW-STEAM. Methods: For DW-STEAM, reference measurements at long diffusion times have significant b0 value, because b = 0 cannot be achieved in practice as a result of the need for signal spoiling. Two strategies for acquiring reference data over a range of diffusion times were considered: constant diffusion weighting (fixed-b0) and constant gradient area (fixed-q0). Fixed-b0 and fixed-q0 were compared using signal calculations for systems with one and two diffusion coefficients, and experimentally using data from postmortem human corpus callosum samples. Results: Calculations of biexponential diffusion decay show that the ADC is underestimated for reference images with b > 0, which can induce an apparent time-dependence for fixed-q0. Restricted systems were also found to be affected. Experimentally, the exaggeration of the diffusion time–dependent effect under fixed-q0 versus fixed-b0 was in a range predicted theoretically, accounting for 62% (longitudinal) and 35% (radial) of the time dependence observed in white matter. Conclusions: Variation in the b-value of reference measurements in DW-STEAM can induce artificial diffusion time dependence in ADC, even in the absence of restriction. Magn Reson Med 79:952–959, 2018.