Acute exercise seems to increase total plasma homocysteine (tHcy); since this variable associated with cardiovascular risk, it is important to understand the determinants of its response to all types of exercise. The aim of this study was to examine the impact of cycling at 2 different rates of muscle contraction on the complete tHcy kinetics. Eight young sedentary males were required to complete 2 isocaloric (400 kcal) acute exercise trials at 50% peak oxygen uptake on separate occasions at 50 or 80 rpm. Blood samples were drawn at different points before (4 h before exercise and immediately before exercise), during (10, 20, 30, 45, and 60 min during exercise), and after exercise (immediately and 19 h after exercise). Dietary and lifestyle factors were controlled during the research. Maximum tHcy occurred during exercise for both conditions (50 rpm: 11.4 ± 2.7 μmol·L; 80 rpm: 10.8 ± 3.2 μmol·L). From this point onwards tHcy declined until the cessation of exercise and continued descending below pre-exercise values at 19 h postexercise (p < 0.05). No hyperhomocysteinemia were observed at any sampling point in both trials. In conclusion, the different muscular contraction frequency during exercise has no impact on tHcy during an acute bout of exercise in sedentary individuals, when at least 400 kcal are spent during exercise and the nutritional status for folate, B, and B is adequate. This information is relevant to further inform healthy exercise prescription, not only in terms of duration and intensity of exercise, but also taking into account frequency of contraction.