Diffusion tensor imaging (DTI) has been used extensively both clinically [1] and for research [2] to map white matter fiber tracts in the central nervous system (CNS). In this application DTI takes advantage of the anisotropic structure of axons, which permits faster diffusion of water molecules parallel to and slower diffusion perpendicular to the axon. DTI has also been applied, although to a much lesser extent, for defining the micro structure of other tissues which also have an inherent structural anisotropy [3]. For example, skeletal muscles consist of bundles of individual muscle fibers, which are in turn comprised of multiple parallel protein bands called myofibrils. This protein configuration and the overall cellular geometry give muscle fibers a highly anisotropic structure. Consequently, DTI can be used to reveal the orientation of muscle fibers [4, 5]. The value of these analyses is derived partly from the observation that the spatial arrangement of muscle fibers with respect to the muscle's line of action is directly related to the muscle's ability to produce force [6, 7]. In particular, two structural phenomena, the pennation angle and muscle fiber length, are integral for assessing the ability of a muscle to generate force. Estimating muscle fiber structure through DTI in fact may be easier than deriving aspects of axonal structure because the muscle fiber diameters, which are approximately 10-90 μm, match the diffusion length within the typical diffusion time of between 10- 50 ms [8, 9]. This could be particularly helpful when assessing changes in muscle fibers as a result of injury or training, where measurements cannot currently be readily made non-invasively over the whole muscle. While there is growing interest in applying these techniques to muscle, data are still lacking regarding the effect of different DTI pulse sequences on the derived muscle fiber length and pennation angle. The application of DTI to muscle presents a number of unique challenges that require adaptation of the twice refocused spin echo (TRSE) MR pulse sequences commonly used in the brain. One factor is the smaller T2 to T1 ratio in muscle versus the white matter in the CNS [10]. One consequence of this is that the signal-to-noise ratio (SNR) in diffusion weighted images is poorer in muscle compared to the brain [11]. An additional challenge, more important to imaging muscle than the CNS, is minimizing the contribution to the image of adipose tissue that surrounds and infiltrates the muscle. Because diffusion weighted images are typically acquired with echo planar imaging (EPI) techniques, the off-resonance protons in fat lead to displacement of the fat in the image so that it overlaps the muscle of interest [5]. The result will be a bias in the estimates of the diffusion coefficients and added variability to the estimates of the eigenvector directions [12, 13]. Because of the much smaller amount of fat in the scalp, this problem is not nearly as significant for imaging the brain. Lastly, localized eddy current-induced fields which result from the prolonged high gradient pulses to encode diffusion can further degrade the geometric fidelity and general image quality [11, 14]. These well recognized challenges have encouraged the exploration of alternative diffusion encoding strategies from the more commonly used TRSE, echo planar acquisitions typically used with DTI. To deal with these challenges some investigators have explored using a single-echo spin echo acquisition similar to the original Stejskal-Tanner technique but with a modified gradient design to reduce eddy current generation [14-16]. Within a TRSE technique alternative bipolar gradient encoding schemes have been investigated to minimize the compromise in image quality and SNR [17]. Bipolar gradient schemes have reduced geometric distortion but at the expense of slightly longer TE than monopolar encoding schemes [18] [14]. A few groups have used a Stimulated Echo Acquisition Mode (STEAM) sequence, wherein the diffusion encoding occurs during the mixing time (TM) [8, 19]. As only T1 recovery occurs during TM, it is possible to increase TM to achieve an adequate b value while simultaneously reducing TE. The lengthened diffusion encoding time also reduces the need for high amplitude gradients, thus reducing the induced eddy currents that spatially distort the images. While in limited studies both techniques have been shown to work in muscle, a comparison between the two pulse sequences, particularly for quantitative fiber tracking applications, is lacking in the literature. Such a comparison will be of value to clinicians and researchers alike to help them optimize or choose the sequence that would work best for their particular application. Previous studies that have evaluated either a STEAM or a TRSE sequence for imaging muscle have used different field strengths (1.5 T and 3.0T), have compared the sequences in-vivo in human muscle, run simulations, or used excised brain tissue [5, 11, 14]. Thus, the purpose of this study was to compare a TRSE DTI sequence to a STEAM DTI sequence on the Vastus Lateralis (VL) of healthy human participants.