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Objective: To compare Dixon-based MRI techniques for intramuscular fat quantification at 3 T with MR spectroscopy (MRS) in vitro and in vivo. Methods: In vitro, two- three- and four-point mDixon (Philips Medical Systems, Best, Netherlands) sequences with 10°, 20° and 30° flip angles were acquired from seven test phantoms with sunflower oil-water percentages of 0-60% sunflower oil and calculated fat-water ratios compared with MRS. In vivo, two- three- and four-point mDixon sequences with 10° flip angle were acquired and compared with MRS in the vastus medialis of nine healthy volunteers (aged 30.6 ± 5.3 years; body mass index 22.2 ± 2.6). Results: In vitro, all mDixon sequences correlated significantly with MRS (r>0.97, p<0.002). The measured phantom percentage fat depended significantly on the flip angle (p⩽0.001) and mDixon sequence (p = 0.005). Flip angle was the dominant factor influencing agreement with MRS. Increasing the flip angle significantly increased the overestimation of the mDixon sequences compared with MRS. In vivo, a significant difference was observed between sequences (p<0.001), with all mDixon sequences overestimating the intramuscular fat content of the vastus medialis muscle compared with MRS. Two-point mDixon agreed best with MRS and had comparable variability with the other mDixon sequences. Conclusion: This study demonstrates that mDixon techniques have good linearity and low variability for use in intramuscular fat quantification. To avoid significant fat overestimation with short repetition time, a low flip angle should be used to reduce T1 effects. Advances in knowledge: This is the first study investigating the optimal mDixon parameters for intramuscular fat quantification compared with MRS in vivo and in vitro. [ABSTRACT FROM AUTHOR]