Running-related limb asymmetries suggest specific sports injuries and recovery circumstances. It is debatable if running speed affected asymmetry, and more research is required to determine how longitudinal bending stiffness (LBS) affected asymmetry. The purpose of this study was to investigate the influence of running velocity and LBS on kinematic characteristics of the hip, knee, ankle, metatarsophalangeal joint (MTP) and the corresponding asymmetry. Kinematic (200 Hz) running stance phase data were collected bilaterally for 16 healthy male recreational runners (age: 23.13 ± 1.17, height: 175.2 ± 1.6 cm, body mass: 75.7 ± 3.6 kg, BMI: 24.7 ± 1.3 kg/m2) running on a force plate at three different velocities (10, 12 and 14 km/h) and three increasing-LBS shoes in a randomized order. The symmetry angle (SA) was calculated to quantify gait asymmetry magnitude at each running velocity and LBS. Changes in running velocity and LBS led to differences in kinematic variables between the hip, knee, ankle and MTP (p < 0.05). Significant changes in SA caused by running velocity were found in the knee flexion angle (p = 0.001) and flexion angle peak velocity (p < 0.001), ankle plantarflexion angle (p = 0.001) and plantarflexion angle peak velocity (p = 0.043) and MTP dorsiflexion angle (p = 0.001) and dorsiflexion angle peak velocity (p = 0.019). A significant change in the SA caused by LBS was found in the MTP dorsiflexion peak angle velocity (p = 0.014). There were interaction effects between running velocity and LBS on the MTP plantarflexion angle (p = 0.033) and plantarflexion angle peak velocity (p = 0.038). These findings indicate the existence of bilateral lower limb asymmetry. Meanwhile, it was proved that running velocity and LBS can influence the asymmetry of lower limb joints. Additionally, there was an interaction between running velocity and LBS on the asymmetry of the lower limb. These findings can provide some information for sports injuries, such as metatarsal stress fractures and anterior cruciate ligament injuries. They can also provide some useful information for running velocities and running shoes.