Whereas quantification of myelin content is crucial for both diagnostic and monitoring purposes in a variety of diseases, a non-invasive tool for myelin quantification in vivo is underdeveloped. Our group has pioneered novel relaxation methods for generating unique types of MRI contrast based on frequency swept radiofrequency (RF) pulses, which have been shown to detect tissue pathologies in various diseases including stroke, Parkinson’s disease and multiple sclerosis. In particular, we have developed a novel rotating frame relaxation method, entitled RAFFn (Relaxation Along a Fictitious Field in the rotating frame of rank n) [1], which produces flexible MRI contrast with low specific absorption rate, and could be particularly sensitive to slow/ultra slow motion in the millisecond time scale. Using animal models in vivo and ex vivo, we have demonstrated that relaxation time constants measured with RAFF4 and RAFF5 are exceptionally sensitive to myelin content and integrity [2, 3], which we attribute to the ability to detect conformational exchange of the methylene groups in myelin sheaths. It had been shown that the high rotating frame configuration of the RAFFn technique is uniquely sensitive to slow motional processes, and provides a better marker of myelin than other MRI methods such as magnetization transfer (MT), T1, T2, T1ρ or T2ρ [3]. In this work, using clinical 3 Tesla Prisma platform we have extended our previous findings where we have shown that the T1r measurements in relapsing–remitting MS (RRMS) patients at 4T [4] could detect abnormalities in the normal appearing white matter (NAWM) of MS patients. We thus embarked on the study of RRMS patients using our novel relaxometry modalities, and we investigated how pathological abnormalities in the white matter of MS patients are detected using T1ρ, T2ρ and RAFF4. We have also compared these MRI metrics with the outcomes of DTI, i.e., mean diffusivity (MD) and fractional anisotropy (FA).All studies were conducted on a 3 T/ 90 cm bore, Siemens Prisma console, 64-channel receive system. Four RRMS individuals (49±10 y/o) and four control subjects (54 ± 5 y/o) participated in this study. For T1ρ, T2ρ and RAFF4 acquisitions, 30 slices were acquired with segmented GRE readout (4 segments), voxel size: 1.6x1.6x3.6 mm3, GRAPPA=3, TE=3.18 ms; TR=2 s. The parameters of the preparation pulses were used as previously described [1, 5-8]. For DTI, parameters were: 128 directions, with 5 additional non-diffusion weighed (b0) images, b-value=1500s/mm2, voxel size 1.8x1.8x1.8mm3, TR=2820 ms, TE=72.6 ms; multi band (MB)=4, 80 slices. Brain segmentation of the high resolution T1-weighted images was carried out with FreeSurfer (FS) version 5.3.0. Rotating frame MRI and DTI metrics were then co-registered to the T1-weighted images, and histograms were extracted from the segmented subcortical WM. Modes of the histograms were then estimated per subject, and compared among subject groups with unpaired two-tailed t-test.Representative parametric maps are shown in Fig. 1, while the results of relaxation and DTI measurements are summarized in Table 1. The T1ρ, T2ρ and RAFF4 relaxation time constants of the WM exhibited significant differences between MS patients and control subjects. Greater differences between MS and controls were observed with T1ρ (p=0.003) and T2ρ (p=0.001) as compared to RAFF4 (p=0.022). Interestingly, no statistically significant differences in the WM between MS patients and controls were detected with MD and FA. Our recent animal studies using healthy rats [3] and mice with mucopolysaccharidosis I (MPS I) [2] have demonstrated that RAFF4 is sensitive predominantly to myelin content and integrity. This was attributed to its enhanced sensitivity to slow / ultra slow motion. Because MS is characterized by several processes such as demyelination, axonal degeneration, gliosis and neuroinflammation, each of which are characterized by different motional regimes, the rotating frame relaxation contrasts have the potential to be sensitive to these pathological processes by “tuning” to the relevant regime of motion.We have used several MRI modalities, i.e., adiabatic T1ρ and T2ρ, RAFF4 and DTI, to investigate their ability to detect abnormalities of the white matter in MS. As opposed to DTI outcomes, each of the rotating frame MRI techniques detected group differences in the white matter of MS patients vs controls, possibly providing sensitivity to different aspects of the MS pathology. In agreement with our previous findings in animal models, RAFFn detected abnormalities in the WM of MS patients which we attribute to the sensitivity of RAFFn to myelin.