Subtalar joint (STJ) dysfunction can contribute to movement disturbances. Vibration energy with color Doppler imaging (VECDI) may be useful for detecting STJ stiffness changes. (1) Support proof-of-concept that VECDI could detect STJ stiffness differences; (2) Establish STJ stiffness range in asymptomatic volunteers; (3) Examine relationships between STJ stiffness and foot mobility; and (4) Assess VECDI precision and reliability for examining STJ stiffness. After establishing cadaveric testing model proof-of-concept, STJ stiffness (threshold units, ΔTU), ankle complex passive range-of-motion (PROM) and midfoot-width-difference (MFWDiff) data were collected in 28 asymptomatic subjects in vivo. Three reliability measurements were collected per variable; Rater-1 collected on all subjects and rater-2 on the first ten subjects. Subjects were classified into three STJ stiffness groups. Cadaveric VECDI measurement intra-rater reliability was 0.80. A significantly lower STJ ΔTU (p = .002) and ankle complex PROM (p < .001) was observed during the screw fixation versus normal condition. A fair correlation (r = 0.660) was observed between cadaveric ΔTU and ankle complex PROM. In vivo VECDI measurements demonstrated good intra-rater (0.76-0.84) versus poor inter-rater (-3.11) reliability. Significant positive correlations were found between STJ stiffness and both dorsum (r = .440) and posterior (r = .390) PROM. MFWDiff exhibited poor relationships with stiffness (r = .103) and either dorsum (r = .256) or posterior (r = .301) PROM. STJ stiffness ranged from 2.33 to 7.50 ΔTUs, categorizing subjects' STJ stiffness as increased (n = 6), normal (n = 15), or decreased (n = 7). Significant ANOVA main effects for classification were found based on ΔTU (p< .001), dorsum PROM (p = .017), and posterior PROM (p = .036). Post-hoc tests revealed significant: (1) ΔTU differences between all stiffness groups (p < .001); (2) dorsum PROM differences between the increased versus normal (p = .044) and decreased (p = .017) stiffness groups; and (3) posterior PROM differences between the increased versus decreased stiffness groups (p = .044). A good relationship was found between STJ stiffness and dorsum PROM in the increased stiffness group (r = .853) versus poor, nonsignificant relationships in the normal (r = -.042) or decreased stiffness (r = -.014) groups. PROM may not clinically explain all aspects of joint mobility. Joint VECDI stiffness assessment should be considered as a complimentary measurement technique.