The goal of the present study was to investigate the effect of positioning a soft flexible tube-based actuator parallel or orthogonal to the principle muscle fibre direction, on measurements of the stiffness of upper trapezius (UT) muscle obtained using magnetic resonance elastography (MRE). The effects of using three different vibration frequencies (60 Hz, 80 Hz and 100 Hz) and studying left and right sides of the body were also investigated. The relevant MRE datasets were acquired on a 1.5 T MRI system using a 2D gradient-echo (GRE) MRE sequence, and corresponding wave images produced using multimodel direct inversion (MMDI) were analysed by two observers using the manual caliper technique. Except for two of the 108 individual datasets, when the agreement was moderate, there was substantial to perfect agreement between wave quality scores obtained by the two observers, with an identical mean value. Similarly, and again with only two exceptions, there was good to excellent agreement between the measurements of UT stiffness obtained by the two observers. UT stiffness values obtained when the acoustic waves were propagating along the principle muscle fibre direction were significantly higher than when the waves were propagating orthogonal to the principle muscle fibre direction at all vibration frequencies (p <
0.005), and only for the former was a significant dispersion effect observed whereby stiffness increased as frequency increased (p <
0.05). No significant asymmetry was observed in measurements of UT stiffness obtained for the left and right sides of the body (p = 0.29). In conclusion, the new soft and flexible tube-based actuator is comfortable and produced very good wave propagation in UT when positioned in either orientation. However, it is recommended for wave propagation to be induced in the principle fibre direction and there was found to be no advantage in using a vibration frequency above 60 Hz.