The study aimed to investigate the physiological and meat quality differences between Non-Ambulatory, Non-Injured (NANI), and without apparent abnormalities (non-NANI) pigs in a commercial slaughterhouse setting, focusing on the impact of stress and health conditions on the overall well-being and meat quality of the animals. A total of 241 surgically castrated crossbred male pigs from Southern Brazil were analyzed, with 131 non-NANI pigs and 110 NANI pigs. Infrared orbital temperature, rectal temperature, hematological parameters, and meat quality measurements were collected. Statistical analysis included ANOVA tests and principal component analysis (PCA). NANI pigs exhibited significantly higher infrared orbital temperatures and rectal temperature (p < 0.01). Hematological analysis revealed higher levels of hemoglobin, hematocrit, and red blood cells in NANI pigs (p < 0.05). White blood cell count and lactate dehydrogenase were significantly elevated in NANI pigs (p < 0.01), indicating potential infections or inflammatory responses. Meat quality parameters showed that NANI pigs had lower pH values, higher luminosity, and increased drip loss (p < 0.01), reflecting poorer water retention and potential muscle glycogen depletion. The study highlights the physiological and meat quality differences between NANI and non-NANI pigs, emphasizing the impact of stress, health conditions, and handling procedures on the animals. Blood biomarkers proved valuable in assessing physiological stress, immune response, and potential health issues in pigs, correlating with meat quality abnormalities. Utilizing these biomarkers as predictive tools can enhance animal welfare practices and contribute to improving meat quality in the swine industry.