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Conflicting results on testicular stiffness in varicocele and the lack of a standardized measurement protocol hinder clinical staging. In this study, we aimed to investigate the effects of probe frequency and region of interest (ROI) size selection on testicular stiffness measurements and to evaluate the testicular parenchymal stiffness and volume changes in patients with varicocele compare with healthy controls under standardized conditions. Shear wave elastography (SWE) was first performed on 62 testes from 31 healthy men, with two probe frequencies (SL10–2 and SL15–4) and four ROI sizes (4, 6, 8, and 10 mm), to assess the impact on SWE measurements. Based on the lowest variability settings, testicular volume and stiffness were compared in 76 patients with varicoceles and 68 healthy controls. Different SWE values were obtained with the two probes and four ROI sizes (p < 0.05). The SL15–4 probe showed lower variability, with the lowest coefficient of variation at the 4 mm ROI. The median testicular SWE value was 2.03 (1.97–2.07) kPa in the control group and 2.01 (1.87–2.07), 1.77 (1.53–1.98), and 1.51 (1.45–1.63) kPa in the Grade I, II, and III varicocele groups, respectively. SWE values were significantly lower in varicocele patients and decreased with increasing grade. The median testicular volumes were 13.08 (12.06–15.70) mL in the control group and 12.26 (10.70–15.15), 13.06 (11.34–14.72), and 11.84 (11.26–12.86) mL in the Grade I, II, and III varicocele groups, respectively. No significant difference in volume was observed between varicocele and control groups (p = 0.387). Probe frequency and ROI size influence testicular SWE measurements, and caution should be exercised when comparing SWE results. Our study showed that testicular stiffness was decreased in patients with varicocele without significant changes in testicular volume, suggesting the presence of subtle structural alterations. SWE may provide complementary information by detecting changes in testicular parenchymal in patients with varicocele.