Purpose: Dual density midsole constructions at the lateral rearfoot and medial midfoot provide opportunities to improve cushioning and stability of running shoes. By similar mechanisms, non-uniform midsole density across the medio-lateral direction at the midfoot to forefoot may allow better negotiation of different loading magnitudes of the medial and lateral midfoot to forefoot during running. Thus, the effect of segmented midsole hardness at the midfoot to forefoot of running shoes on perception and biomechanics was examined. Methods: Four custom-made running shoes featured a three section longitudinal hardness pattern at midfoot to forefoot. The central section as well as the rearfoot section always had consistent medium hardness, whereas medial and lateral sections were systematically softer or harder. A sample of 24 runners participated in visual analogue scale based perception measurements and in recording of in-shoe plantar pressures, ground reaction forces, and multi-segment foot kinematics. Shoe effects were analysed by repeated measures analyses of variance (ANOVA) (p<.05), followed by least significant difference (LSD) and Bonferroni adjusted post-hoc tests (p<.05) for discrete variables. Interaction of shank and foot segments was also analysed using motion-time curves. Results: Runners distinguished midsole hardness at the medial (p D .009) but not at the lateral midfoot to forefoot and indicated a trend (p D .069) preferring softer medial hardness. Plantar peak pressure (medial: p D .006, lateral: p D .000) and relative loads (medial: p D .000, lateral: p D .000) were increased when midsole sections were harder. Ground contact time (p D .045) and maximum loading rate I (p D .016) were higher for shoes having softer medial hardness, while rearfoot (p D .040) and forefoot (p D .001) showed increased maximum ankle eversion for these conditions. Conclusion: Segmented running shoe midsole hardness across the medio-lateral direction of the midfoot to forefoot influences subjective comfort and biomechanical forefoot stability. Findings allow systematic approaches to improve both features, thereby creating enhanced running footwear.