Lactate dehydrogenase is a well-studied oxidoreductase with profound implications in diseases like cancer and is a nonspecific marker of tissue damage. In vitro investigations into the mechanism of lactate dehydrogenase action reported herein are based on our group’s earlier reported thermodynamic calculations. While the reverse (pyruvate to lactate) reaction is thermodynamically favourable, the forward (lactate to pyruvate) reaction is highly unfavourable at physiological conditions. Herein, we studied the rates of NADH utilization and NADH formation, in the reverse and forward reactions, respectively, at varying pH conditions in three different buffer systems. NADH consumption rates were high, in the range of 2000-3000 M -1 s -1 and pH 7.4 was ideal for the pyruvate-lactate reduction reaction. However, in the forward reaction, the NADH formation rates were low (50 to < 300 M -1 s -1), if at all! Strikingly, inclusion of lactate dehydrogenase into liver tissue lysate caused a rapid decline of NADH formation rates. Fenton reaction using Fe 2+ and hydrogen peroxide catalyzed NADH formation, but again, addition of lactate dehydrogenase drastically decreased Fenton reaction-mediated NADH formation. Murburn, via one-electron oxidation of guaiacol by horseradish peroxidase, rapidly accelerated NADH formation rate in the range of 300-800 M -1 s -1; but consistently, addition of lactate dehydrogenase in the reaction significantly lowered the NADH formation rate. Another reaction was carried out with potassium superoxide and lactate to track NADH formation from NAD + and again, addition of both D- and L-lactate dehydrogenase led to a decline in NADH formation rates in the forward reaction. Gas-chromatography mass spectrometry and FT-IR results confirmed the superoxide-assisted oxidation of lactate, while addition of lactate dehydrogenase into this reaction once again suppressed lactate oxidation to pyruvate. These findings have phenomenal implications in the reaction mechanism of lactate dehydrogenase and the cellular processes that are linked to lactate dehydrogenase action.