The present study examined the impact of single sessions of graded treadmill exercise on lactate dehydrogenase (LD) in young healthy athletes. A cohort of 20 physically fit male athletes aged 23.75 ± 1.45 years were randomly chosen and underwent single sessions of 5 min each of high-intensity exercise (HIE) at 77–95% of maximal heart rate (HRmax), moderate-intensity exercise (MIE) at 64–76% of HRmax, and low-intensity exercise (LIE) at 57–63% of HRmax, separated by a one-week washout period. LD levels were measured in blood samples taken before, immediately after, and 10 and 20 min after each form of exercise. LD levels differed significantly across intensity (ES = 0.34; p = 0.007) and time points (ES = 0.58; p = 0.000), as determined by repeated measures ANOVA. A post-hoc analysis with Bonferroni adjustment indicated that HIE resulted in a significant rise in LD levels compared to moderate (p = 0.011) and low intensity (p = 0.002) activities. Furthermore, post-hoc analysis revealed a significant rise in LD levels immediately post-exercise (p = 0.002), followed by a decline at 10 min post-exercise, ultimately returning close to baseline levels after 20 min. The study highlights the role of exercise intensity in regulating LD levels, providing insights into metabolic responses and potential applications in exercise prescription and rehabilitation protocols.