In this study we performed laboratory treadmill protocols of increasing load. Heart rate was continuously recorded and blood lactate concentration was measured for determination of lactate threshold by means of LTD-max and LT4.0 methods.Our results indicate that the shape of heart rate performance curve (HRPC) during incremental testing depends on the applied exercise protocol (change of initial speed and the step of running speed increase, with the constant stage duration). Depending on the applied protocol, the HRPC can be described by linear, polynomial (S-shaped), and exponential mathematical expression.We presented mathematical procedure for estimation of heart rate threshold points at the level of LTD-max and LT4.0, by means of exponential curve and its relative deflection from the initial trend line (tangent line to exponential curve at the point of starting heart rate). The relative deflection of exponential curve from the initial trend line at the level of LTD-max and/or LT4.0 can be defined, based on the slope of the initial trend line. Using originally developed software that allows mathematical analysis of heart rate-load relation, LTD-max and/or LT4.0 can be estimated without direct measurement of blood lactate concentration.