Decreased tendon stiffness is considered to be a factor in the progression of tendon injury, and research is ongoing to develop methods for quantitatively assessing tendon stiffness. Strain ratio (SR) measurement using the strain elastography function of an ultrasound diagnostic device can noninvasively measure the stiffness of living tissue based on the ratio of strain values between the acoustic coupler and the measured material, and thus can be used to assess the stiffness of tendons. However, the reliability of such measurements remains an issue. Therefore, the purpose of this study was to clarify the influence of preloading on the SR measurement of a phantom mimicking a tendon with decreased stiffness. Independent inclusions with stiffness values of 100, 200, and 400 kPa were placed in the phantom and measured. Preloading was set to 0.5, 1.0, and 1.5 N, and the SR was measured by applying an mechanically controlled additional load of 3.0 N. Taking the average of three measurements for each inclusion as a representative value, three sets of measurements were performed on different days for each preloading condition. The intraclass correlation coefficient (ICC) was used to examine the intra-observer reliability between days. In addition, the SR among preloading conditions was compared. ICC (1, 1) was 0.99 for all preloading conditions, indicating excellent reliability. The comparison among preloading conditions showed that the SR significantly decreased at a preloading of 1.5 N compared with 0.5 N for all inclusions. These results suggest the importance of controlling preloading during SR measurements. © 2024, Japan Soc. of Med. Electronics and Biol. Engineering. All rights reserved.