Our investigation elucidated the effects of severe temperature fluctuations on cellular and physiological responses in beef cattle. Eighteen Red Angus beef steers with an average body weight of 35124.5 kg were divided into three treatment groups: 1) Control (CON), exposed to a temperature-humidity index (THI) of 42 for 6 h without any temperature changes; 2) Transport (TP), subjected to a one-mile trailer trip with a THI of 42 for 6 h; and 3) Temperature swing (TS), exposed to a one-mile trailer trip with a THI shift from 42 to 72-75 for 3 h. Our findings indicate that TS can induce thermal stress in cattle, regardless of whether the overall temperature level is excessively high or not. Behavioral indications of extreme heat stress in the cattle were observed, including extended tongue protrusion, reduced appetite, excessive salivation, and increased respiratory rate. Furthermore, we observed a pronounced overexpression (P<0.05) of heat shock proteins (HSPs) 20, 27, and 90 in response to the TS treatment in the longissimus muscle (LM). Alterations in signaling pathways associated with skeletal muscle growth were noted, including the upregulation (P<0.01) of Pax7, Myf5, and myosin heavy chain (MHC) isoforms. In addition, an increase (P<0.05) in transcription factors associated with adipogenesis was detected (P<0.05), such as PPAR gamma, C/EBP alpha, FAS, and SCD in the TS group, suggesting the potential for adipose tissue accumulation due to temperature fluctuations. Our data illustrated the potential impacts of these temperature fluctuations on the growth of skeletal muscle and adipose tissue in beef cattle. Our study demonstrated how temperature swings can induce thermal stress in beef cattle, leading to observable behavioral and physiological changes. Our findings revealed the impact of severe temperature fluctuations on the signaling pathways involved in skeletal muscle growth and the potential for adipose tissue accumulation. While gaining insight into the cellular and physiological responses of beef cattle to temperature variations, elucidated on the complex interplay between environmental factors and animal productivity. Lay Summary In this study, we investigated the effects of severe temperature fluctuations on beef cattle and their cellular and physiological responses. Our findings demonstrate that even moderate temperature swings can cause thermal stress in cattle, leading to observable behavioral signs such as extended tongue protrusion, reduced appetite, excessive salivation, and increased respiratory rate. We also observed a significant increase in the expression of heat shock proteins (HSPs), which protect cells from stress, indicating their importance as early responders to temperature fluctuations. Furthermore, we examined the signaling pathways involved in skeletal muscle growth and found that severe temperature fluctuations can stimulate the upregulation of myogenic regulatory factors and myosin heavy chains. These changes suggest an increased demand for muscle contractile properties and hyperplasia during temperature challenges. In addition, our study revealed alterations in transcription factors associated with adipogenesis, such as PPAR gamma and C/EBP alpha, indicating the potential for adipose tissue accumulation in response to temperature fluctuations.