In asphalt pavement structure design, traffic axle loads and pavement layer temperatures are crucial factors affecting fatigue damage calculations. To investigate the differences in fatigue damage calculations caused by different characterizations of traffic axle loads and temperature, fatigue damage calculations were conducted under equivalent standard axle loads (ESALs), axle load spectra (ALS), constant temperatures, and seasonal temperature variations using the field data from an expressway in Shandong Province, China under seven calculation plans. The results indicated: (1) the annual traffic composition is dominated by vehicle Type 9, with a proportion of about 43% in all the vehicle types, and its load level is also high, with a proportion about of 80% in the heavy load interval at all axle types; (2) The ESALs method underestimates the actual fatigue damage incurred in asphalt pavement by 6.04 times, with an accumulated damage of 2.34 × 10−9 (ESALs), 1.69 × 10−8 (ALS), respectively; (3) The fatigue damage results from a single month with consistent temperature showed similar trends, with an accumulated damage of 1.50 × 10−5, 9.07 × 10−5, respectively; (4) The cumulative fatigue damage calculated using the ALS method across the four seasons, respectively, is 6.51, 5.88, 6.42, and 4.60 times that of the fatigue damage calculated using the ESALs method. Although the ratio of fatigue damage between the two characterizations of traffic axle loads remains consistent, which is 6.04, the fatigue damage calculation that accounts for temperature variations can reveal seasonal trends in fatigue damage development. Based on the axle load spectra and considering temperature variations, fatigue damage calculation will be more closely related to the actual service state of asphalt pavement. These research findings provide insights for estimating asphalt pavement fatigue damage to some extent.