Blood samples were obtained from 16 high-risk heifers; eight were newly arrived from a 40 h road trip (0 days post-arrival (DPA)), whereas the other eight heifers had been in the feedlot at 25 DPA. Both groups were transported from the southeast tropical region of Mexico to a feedlot in the northeast and were sampled on the same day. The complete blood count, blood chemistry, and cytokine gene expression were analyzed. Gene expression was analyzed using specific primers to amplify and quantify the cDNA reverse transcribed from the mRNA transcripts for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2. Higher values for hematocrit (p = 0.029), hemoglobin (p = 0.002), eosinophils (0.029), albumin (p = 0.014), alanine aminotransferase (p = 0.004), bilirubin (p = 0.003), cholesterol (p = 0.014), and cortisol (p = 0.051) were observed in the 0 DPA group than the 25 DPA group. In the electrophoresis of TNF-α amplification products, two non-specific bands were observed in the 0 DPA group. These bands were sequenced, and BLAST analysis suggested that they corresponded to bovine lymphotoxin and have not been reported previously related to stress. The TNF-α expression level was higher (p = 0.001) in the 25 DPA group than the 0 DPA group according to the semi-quantitative expression analysis. This may indicate a persistent inflammatory process that could be related to trauma and disease, which can negatively impact their subsequent health and growth performance. In conclusion, homeostatic disruption was apparent in the 0 DPA heifers, which showed higher cortisol and reductions in TNF-α levels and stress-induced bovine lymphotoxin (SIBL) co-expression.