The aim of this study was to investigate the expression of cardiac strain and damage in 18 male marathoners with average age of 52.8 ± 5.0 years running at a 308 km ultra-marathon. Blood samples were collected at pre-race, 100 km, 200 km and 308 km check points for the analysis of cardiac muscle injury markers, creatine kinase (CK), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTnI) and cardiac muscle strain marker, N-terminal pro-brain natriuretic peptide (NT-proBNP). The CK levels increased 1127.2 ± 507.9 IU/L, 5133.8 ± 2492.7 IU/L and 4958.4 ± 2087.9 IU/L at 100 km, 200 km and 308 km, respectively, compared to the pre-race levels. The CK-MB levels increased 20.2 ± 11.2 ng/mL, 73.3 ± 35.6 ng/mL and 68.6 ± 42.6 ng/mL at 100, 200 and 308 km, respectively, compared to the pre-race levels. The CK-MB/CK ratio showed that the CK-MB mass index was within the normal range (<2.5%) at 100 km, 200 km and 308 km. The cTnI levels showed no significant difference in all check points. The NT-proBNP levels increased 146.55 ± 92.7 pg/mL, 167.95 ± 111.9 pg/mL and 241.23 ± 121.2 pg/mL at 100, 200 and 308 km, respectively, compared to the pre-race levels. The normal CK-MB mass index (<5.0 ng/mL) and the absence of an increase in the cTnI levels during the 308 km ultra-marathon suggested that no myocardial injury despite an elevation in CK-MB. The increase in NT-proBNP levels probably resulted from continuous hemodynamic cardiac stress and represents a transient physiological myocardial protective response.