PurposeTo minimize the known biases introduced by fat in rapid T1 and T2 quantification in muscle using a single‐run magnetic resonance fingerprinting (MRF) water–fat separation sequence.MethodsThe single‐run MRF acquisition uses an alternating in‐phase/out‐of‐phase TE pattern to achieve water–fat separation based on a 2‐point DIXON method. Conjugate phase reconstruction and fat deblurring were applied to correct for B 0 inhomogeneities and chemical shift blurring. Water and fat signals were matched to the on‐resonance MRF dictionary. The method was first tested in a multicompartment phantom. To test whether the approach is capable of measuring small in vivo dynamic changes in relaxation times, experiments were run in 9 healthy volunteers; parameter values were compared with and without water–fat separation during muscle recovery after plantar flexion exercise.ResultsPhantom results show the robustness of the water–fat resolving MRF approach to undersampling. Parameter maps in volunteers show a significant (P < .01) increase in T1 (105 ± 94 ms) and decrease in T2 (14 ± 6 ms) when using water–fat‐separated MRF, suggesting improved parameter quantification by reducing the well‐known biases introduced by fat. Exercise results showed smooth T1 and T2 recovery curves.ConclusionWater–fat separation using conjugate phase reconstruction is possible within a single‐run MRF scan. This technique can be used to rapidly map relaxation times in studies requiring dynamic scanning, in which the presence of fat is problematic.