Purpose Most athletes experience short-term training cessation because of illness, injury, post-season vacation, or other reasons. Passive muscle stiffness is a potential risk factor for a sprint-type hamstring strain injury, but limited information is available about the effect of short-term training cessation on passive muscle stiffness. The present study aimed to identify whether and how passive muscle stiffness of the biceps femoris long head (BFlh) would vary due to 2 weeks of training cessation in sprinters. Methods Passive BFlh shear-wave speed (a proxy for stiffness) was measured using ultrasound shear-wave elastography in 28 male sprinters, before and after 2 weeks of intervention. During the 2 weeks, the participants in the training-cessation group (n = 14) were allowed to maintain their normal daily activities but not to perform any physical training, including stretching and resistance exercises. The participants in the training continuation group (n = 14) performed the training (including maximum speed sprint, plyometric, and weight training) prescribed by their coaches 5 days per week. Results In the training-cessation group, passive BFlh shear-wave speed increased after the 2 weeks of training cessation (4.75 +/- 0.77 to 5.00 +/- 0.88 m/s, P < 0.001). In contrast, there was no significant difference before and after the 2 weeks of training continuation (4.90 +/- 0.85 to 4.93 +/- 0.85 m/s, P = 0.521). Conclusions The present findings indicate that muscles stiffen by training cessation in sprinting athletes.