Perpetual pavements, if properly designed and rehabilitated, last longer than 50 years without major structural rehabilitation. The fatigue endurance limit (EL) is a key parameter for designing perpetual pavements to mitigate bottom-up fatigue cracking. The endurance limit has not been completely implemented in the Mechanistic Empirical Pavement Design Guide software, currently known as AASHTOWare Pavement ME Design software. This study was conducted as part of the National Cooperative Highway Research Program (NCHRP) project 9-44A to develop a framework and mathematical methodology to determine the EL for hot mix asphalt (HMA) using the uniaxial tension-compression fatigue test. In this unique procedure, the EL is defined as the allowable tensile strains at which a balance takes place between the fatigue damage during loading and the healing during rest periods between loading pulses. The viscoelastic continuum damage model was applied into the data analysis. This study also included the development of a uniaxial fatigue test method and the associated data acquisition computer programs to conduct the test with and without rest period. The laboratory testing program consisted of dynamic modulus testing to estimate the viscoelastic properties of the asphalt mixtures and a uniaxial fatigue test experiment conducted with and without rest periods. Five factors that affect the fatigue and healing behavior of asphalt mixtures were evaluated: asphalt content, air voids, temperature, rest period, and tensile strain. On the basis of the test results, a pseudo stiffness ratio (PSR) regression model was developed as a function of the five factors and the number of loading cycles. The EL was defined when PSR is equal to 1.0 (net damage is equal to zero). The results from the sensitivity analysis showed rational relationships between the EL and investigated factors. The EL value was observed to increase by increasing temperature, asphalt content, and rest periods, whereas it decreased when air voids increased.