To evaluate heart rate as a patient-related confounder in a commonly applied T2 balanced steady-state free precession (bSSFP) mapping sequence used for myocardial tissue characterization.Materials and Methods: This retrospective analysis included prospectively (from December 2013 to November 2021) acquired cardiac MRI (1.5 T) datasets with T2 bSSFP mapping from 69 healthy volunteers. Phantom studies and Bloch simulations were performed with heart rates of 60–130 beats per minute and different resting periods (three, six, or nine R-R intervals). Sequence parameters (repetition time, echo time, flip angle, echo train length) were matched across volunteer, phantom, and simulation measurements. Reference values covered clinically relevant T1 and T2 properties found in native myocardium (short, 1041 and 44 msec; medium, 1293 and 43 msec; long, 1534 and 40 msec). A mixed linear model assessed the effect of heart rate on T2 values in volunteer measurements.Results: The study included 69 healthy volunteers (median age, 34 years; 44 female and 25 male). Heart rate influenced T2 values acquired with three R-R resting periods (r = −0.38, P =.002; linear regression slope, −0.7 msec/10 beats per minute [95% CI: −1.2, −0.1]). In simulation and phantom measurements, T2 values acquired with three R-R resting periods strongly correlated with heart rate, irrespective of myocardial T1 and T2 properties (r ≤ −0.88; P <.01 for all measurements). Heart rate dependency was reduced with increased resting periods in simulations and phantom measurements. Short myocardial T1 and T2 values derived from T2 bSSFP with nine R-R resting periods were not dependent on heart rate (r = −0.41; P =.33).Conclusion: T2 bSSFP with three R-R resting periods underestimates T2 values with increasing heart rates. Use of longer resting periods with T2 bSSFP mapping sequences reduced heart rate dependency.