Physical exercise triggers efferent cardiac sympathetic activation. Here, we tracked the spatiotemporal properties of cardiac repolarization on a beat-to-beat basis throughout a standardized exercise test and hypothesized a detectable change at the point of the anaerobic threshold (AT). The study included 20 healthy adults (age 35.3 ± 6.7 yr) undergoing a standardized incremental exercise test on a cycle ergometer. During exercise testing, high-resolution (2000 Hz) ECG monitoring in Frank lead configuration was performed. Three-dimensional beat-to-beat repolarization instability (dT°) was assessed by a novel vector-based method according to a previously published technology. In parallel, the lactate threshold (LT) was detected according to Dickhuth and Mader. We could identify a characteristic pattern of dT° signal during exercise testing. With increasing physical activity, dT° increased concordantly to heart rate. At an average of 164 ± 38 W, dT° and heart rate abruptly showed a discordant behavior, characterized by a transient drop of dT°. The maximal discordance between dT° and heart rate was defined as ATdT° and highly significantly correlated with LTDickhuth (r = 0.841, P < 0.001) and LTMader (r = 0.819, P < 0.001), which were at 156 ± 39 and 172 ± 46 W, respectively. The characteristic of dT° could not be provoked by fast atrial pacing in the absence of exercise. Repolarization instability shows a characteristic pattern during standardized exercise in healthy individuals that allows for a noninvasive estimation of AT.