PurposeDouble‐wave diffusion experiments offer the possibility of probing correlation between molecular diffusion at multiple time points. It has recently been shown that this technique is capable of measuring the exchange of water across cellular membranes. The aim of this study was to investigate the effect of macroscopic tissue anisotropy on the measurement of the apparent exchange rate (AXR) in multicompartment systems.MethodsAXR data were collected from yeast and perfusion‐fixated brain tissue at high angular resolution on a preclinical imaging system. The AXR was expanded for anisotropic systems by calculating scalar AXR values along the principal directions of the diffusion tensor.ResultsIn yeast, both the AXR and diffusivity were rotational invariant, whereas in fixated brain tissue, the measured AXR was sensitive to the orientation of anisotropic structures. AXR, especially in white matter, was robustly estimated along the first and second principal directions of the diffusion tensor, but increasing noise was seen in the AXR estimates along the third principal direction of the diffusion tensor.ConclusionOur results indicate that tissue anisotropy must be considered for AXR estimates in complex biological systems. Magn Reson Med 72:756–762, 2014. © 2013 Wiley Periodicals, Inc.