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This single-blind and cross-sectional study evaluated the role of Rho-kinase (ROCK) as a biomarker of the cardiovascular remodelling process assessed by echocardiography in competitive long-distance runners (LDRs) during the training period before a marathon race. Thirty-six healthy male LDRs (37.0 ± 5.3 years; 174.0 ± 7.0 height; BMI: 23.8 ± 2.8; <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mover accent="true"><mi mathvariant="normal">V</mi><mo>˙</mo></mover></semantics></math></inline-formula> O₂-peak: 56.5 ± 7.3 mL·kg⁻¹·min⁻¹) were separated into two groups according to previous training level: high-training (HT, <i>n</i> = 16) ≥ 100 km·week⁻¹ and low-training (LT, <i>n</i> = 20) ≥ 70 and < 100 km·week⁻¹. Also, twenty-one healthy nonactive subjects were included as a control group (CTR). A transthoracic echocardiography was performed and ROCK activity levels in circulating leukocytes were measured at rest (48 h without exercising) the week before the race. The HT group showed a higher left ventricular mass index (LVMi) and left atrial volume index (LAVi) than other groups (<i>p</i> < 0.05, for both); also, higher levels of ROCK activity were found in LDRs (HT = 6.17 ± 1.41 vs. CTR = 1.64 ± 0.66 (<i>p</i> < 0.01); vs. LT = 2.74 ± 0.84; (<i>p</i> < 0.05)). In LDRs a direct correlation between ROCK activity levels and LVMi (r = 0.83; <i>p</i> < 0.001), and LAVi (r = 0.70; <i>p</i> < 0.001) were found. In conclusion, in male competitive long-distance runners, the load of exercise implicated in marathon training is associated with ROCK activity levels and the left cardiac remodelling process assessed by echocardiography.