Description: Purpose The purpose of this study is to develop double diffusion encoding (DDE) MRI methods for clinical use. Microscopic diffusion anisotropy measurements from DDE promise greater specificity to changes in tissue microstructure compared with conventional diffusion tensor imaging, but implementation of DDE sequences on whole-body MRI scanners is challenging because of the limited gradient strengths and lengthy acquisition times. Methods A custom single-refocused DDE sequence was implemented on a 3T whole-body scanner. The DDE gradient orientation scheme and sequence parameters were optimized based on a Gaussian diffusion assumption. Using an optimized 5-min DDE acquisition, microscopic fractional anisotropy (μFA) maps were acquired for the first time in multiple sclerosis patients. Results Based on simulations and in vivo human measurements, six parallel and six orthogonal diffusion gradient pairs were found to be the minimum number of diffusion gradient pairs necessary to produce a rotationally invariant measurement of μFA. Simulations showed that optimal precision and accuracy of μFA measurements were obtained using b-values between 1500 and 3000 s/mm 2 . The μFA maps showed improved delineation of multiple sclerosis lesions compared with conventional fractional anisotropy and distinct contrast from T 2 -weighted fluid attenuated inversion recovery and T 1 -weighted imaging. Conclusion The μFA maps can be measured using DDE in a clinical setting and may provide new opportunities for characterizing multiple sclerosis lesions and other types of tissue degeneration. Magn Reson Med, 2017. © 2017 International Society for Magnetic Resonance in Medicine.