Background: Mules are used worldwide for agrarian purposes such as plowing and harvesting crops. In this study, mules were used for plowing and cultivation. While mules are believed to be less susceptible to diseases and fatigue than horses, no scientific investigation has been conducted to support this belief. The objective was to assess effect of work-associated fatigue on hematological and biochemical parameters in working mules in Mexico. Methodology: Six healthy mules 6.5 to 23 years, 3.5 BCS, were studied. Three blood samples/day were taken at the beginning and end of the work season. One sample taken at the beginning of the day, middle, and end (3 hours apart). Biochemical parameters, including the concentrations of albumin, globulin, total protein, glucose, alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), total bilirubin, lactate, creatinine kinase (CK), urea, creatinine, calcium, sodium, potassium, total carbon dioxide (tCO2), and al-bumin-to-globulin ratio, were determined using the veterinary Skyla-VB1 automated analyzer (Lite-On Technology Corporation, Hsinchu-Taiwan), and blood lactate concentration using the handheld Lactate Scout Vet. Results: Significant increases in concentrations AST (P < 0.001), BUN (P < 0.001), calcium (P = 0.002), creatine phosphokinase CPK U/L (P < 0.001), tCO2 (P = 0.006), and urea (P < 0.01), were observed at the end of the work season. However, sodium, potassium, and chloride were significantly decreased at the end of the work season. No treatment or time-by-treatment interaction effect was observed for ALP, creatinine, globulin, or the albu-min-to-globulin ratio. Conclusions: The significant decrease in mineral and electrolytes (Na, K, and Cl) at the end of work season could suggest change in electrolytic balance during the strenuous plowing season. A higher concentration of tCO2 at end of the season is a good indicator of metabolic acid-base disturbances. Results indicate use of mules for plowing fields disrupts the mules' micromineral and electrolyte profiles. Increased CPK levels demonstrate that the workload was excessive. This study furthers our understanding of biochemical responses in mules.