Passive stiffness, a biomechanical parameter, has a potential influence on running economy, thus playing a pivotal role in performance. This study aimed to quantify passive stiffness of the Achilles tendon and gastrocnemius muscle using myotonometry throughout an incremental running protocol. Twenty-one male participants underwent a multistage incremental test (Bordeaux test) on a treadmill until exhaustion. Passive stiffness of the gastrocnemius muscle and Achilles tendon was measured using aMyotonPRO device. Measurements were taken before, during, and after the incremental test. Our findings indicated that passive stiffness of the medial gastrocnemius decreased at rest between prerun and postrun assessments (-20.12N·m-1, P = .012). Furthermore, during the test, stiffness increased at low intensity (at 50% of maximal aerobic speed: +104.8N·m-1, P = .042), returning to baseline values as intensity increased. Throughout an incremental running protocol, both Achilles tendon and gastrocnemius muscle stiffness initially increased and then decreased with escalating percentages of maximal aerobic speed. A deeper understanding of stiffness in running can inform more effective recommendations for runners' strengthening and training.