During marketing, cattle may be exposed to periods of water deprivation. The impact of water and feed access and health status on the physiological well-being and carcass characteristics of Holstein slaughter cows during preslaughter marketing was studied through analysis of serum components, BW loss percentage, and fresh meat composition. Ninety-one multiparous Holstein cows (609 ± 89 kg mean BW, 2.9 ± 0.5 mean BCS, varying stage of lactation) were purchased over 3 wk in 3 groups (n = 31, 29, and 31) at a terminal market in central Wisconsin. Each cow was screened to determine health status (sick or not sick) and randomly assigned to 1 of 3 water and feed withdrawal treatment pens (AL, ad libitum access to water for 36 h; 18H, 18 h of ad libitum access to water followed by 18 h of water withdrawal; 36H, 36 h of water withdrawal; all 3 treatments included 36 h of feed withdrawal) in a randomized complete block arrangement with repeated measures for serum components. Blood samples were collected by tail venipuncture at 0, 9, 18, 27, and 36 h of each treatment. Ambient temperatures were 1.9 ± 6.2°C during the trial period, which occurred over a 3-wk period in March and April 2007 near Arlington, WI. No difference (P > 0.05) was observed in mean serum cortisol in AL (18.41 ± 2.17 ng/mL) or 36H (22.98 ± 2.17 ng/mL). Mean serum glucose was greater (P < 0.05) in 36H pens (78.15 ± 0.77 mg/dL) than AL (75.91 ± 0.77 mg/dL). Mean serum creatinine was greater (P < 0.05) in 36H pens (0.71 ± 0.03 mg/dL) than AL (0.60 ± 0.03 mg/dL). The 36H pens also displayed increased (P < 0.05) serum albumin, anion gap, Ca, Cl, Na, cholesterol, and aspartate aminotransferase over AL. Greater (P < 0.05) mean percentage BW loss was observed in 36H pens (5.2 ± 0.6%) than AL (3.1 ± 0.6%). Mean muscle protein (%) was greater (P < 0.05) in 36H (22.2 ± 0.4%) than 18H (21.3 ± 0.4%). Mean muscle moisture (%) was greater (P < 0.05) in AL and 18H (75.3 ± 0.4% and 75.2 ± 0.4%) than 36H. Mean 24-h pH values were 5.92 (AL), 5.92 (18H), and 5.81 (36H; SE = 0.04) and were not different (P < 0.05). Observed pH and color values indicated a borderline dark-cutter state across all cattle in the study, regardless of water and feed access treatment. Based on these results, water and feed withdrawal in lairage should not exceed 18 h during the marketing of Holstein slaughter cows acclimated to springtime conditions to maintain BW, serum component concentrations, and fresh meat composition.