Objective To study the effects of prolonged strenuous military training on plasma biological markers and provide evidence of screening subclinical exertional rhabdomyolysis (ERM) and preventing occurrence of complication. Methods Twenty-three soldiers had a march in battle gear; and heart rate (HR), blood lactic acid (LA), blood potassium, blood sodium, plasma crystal osmotic pressure, creatine kinase (CK), myoglobin (Mb) and cell-free plasma DNA (cf-DNA) were determined on pre-training at rest, immediately post-training and in the next morning at rest. Results Blood potassium, blood sodium and plasma crystal osmotic pressure post-training slightly rose (P＜0.05 or P＜0.01) and recovered in next morning (P>0.05). CK and Mb post-training increased by 58.4% (P＜0.05) and 200% (P＜0.01),respectively, while in next morning, CK level reached 5 times that pre-training (P＜0.01) and Mb reduced significantly (P＜0.01) but was still higher than pre-training (P＜0.05). Cf-DNA elevated obviously post-training (P＜0.01) and restored to pre-training level in next morning (P>0.05). Conclusions Prolonged strenuous military training usually causes subclinical ERM and medical supervision should be enhanced before and after training. It is significant to monitor plasma biological markers for early detection of ERM and prevention of ERM complication.