Key points Strenuous endurance exercise induces transient functional and biochemical cardiac perturbations that persist for 24–48 h. The magnitude and time‐course of exercise‐induced reductions in ventricular function and increases in cardiac injury markers are influenced by the intensity and duration of exercise. In a human experimental model, exercise‐induced reductions in ventricular strain and increases in cardiac troponin are greater, and persist for longer, when exercise is performed within the heavy‐ compared to moderate‐intensity exercise domain, despite matching for total mechanical work. The results of the present study help us better understand the dose–response relationship between endurance exercise and acute cardiac stress/injury, a finding that has implications for the prescription of day‐to‐day endurance exercise regimes. Abstract Strenuous endurance exercise induces transient cardiac perturbations with ambiguous health outcomes. The present study investigated the magnitude and time‐course of exercise‐induced functional and biochemical cardiac perturbations by manipulating the exercise intensity–duration matrix. Echocardiograph‐derived left (LV) and right (RV) ventricular global longitudinal strain (GLS), and serum high‐sensitivity cardiac troponin (hs‐cTnI) concentration, were examined in 10 males (age: 27 ± 4 years; V̇O2, peak : 4.0 ± 0.8 l min−1) before, throughout (50%, 75% and 100%), and during recovery (1, 3, 6 and 24 h) from two exercise trials. The two exercise trials consisted of 90 and 120 min of heavy‐ and moderate‐intensity cycling, respectively, with total mechanical work matched. LVGLS decreased (P < 0.01) during the 90 min trial only, with reductions peaking at 1 h post (pre: −19.9 ± 0.6%; 1 h post: −18.5 ± 0.7%) and persisting for >24 h into recovery. RVGLS decreased (P < 0.05) during both exercise trials with reductions in the 90 min trial peaking at 1 h post (pre: −27.5 ± 0.7%; 1 h post: −25.1 ± 0.8%) and persisting for >24 h into recovery. Serum hs‐cTnI increased (P < 0.01) during both exercise trials, with concentrations peaking at 3 h post but only exceeding cardio‐healthy reference limits (14 ng l−1) in the 90 min trial (pre: 4.2 ± 2.4 ng l−1; 3 h post: 25.1 ± 7.9 ng l−1). Exercise‐induced reductions in ventricular strain and increases in cardiac injury markers persist for 24 h following exercise that is typical of day‐to‐day endurance exercise training; however, the magnitude and time‐course of this response can be altered by manipulating the intensity–duration matrix.