Dairy cows that are restricted from lying down have a reduced ability to sleep. In other species, sleep loss is a key risk factor for disease, mediated by changes in metabolic and inflammatory responses. The cumulative effect of lying and sleep deprivation on cow health is unknown. The objective was to determine the effects of lying and sleep deprivation on metabolic and inflammatory responses of dairy cows. Data were collected from 8 multiparous and 4 primiparous lactating cows (199 ± 44 d in milk, 77 ± 30 d pregnant; mean ± standard deviation) enrolled in a study using a crossover design. Each cow was exposed to 2 treatments meant to induce sleep loss: (1) human disturbance (imposed by researchers making noise or physical contact when the cow's posture suggested sleep) and (2) lying deprivation (imposed by a wooden grid placed on the pen floor). Cows experienced a 24-h baseline period (d -1) followed by a 24-h treatment period (d 0), with a 12-d washout period between treatments. Baseline and treatment periods were imposed from 2100 to 2059 h. Cows were housed in individual pens during the acclimation period (d -3 and -2), d -1, and d 0. Nonesterified fatty acid and glucose concentrations were measured at 0300, 0900, 1500, and 2059 h on d -1 and 0. Proinflammatory cytokine mRNA [tumor necrosis factor (TNF), interleukin-1B (IL1B), and interleukin-6 (IL6)] abundance in whole-blood leukocytes, both nonstimulated and stimulated with lipopolysaccharide, were assessed at 2059 h on d -1 (end of baseline) and d 0 (end of treatment). Nonesterified fatty acids and glucose varied by time of day but were not affected by treatment or day. The abundances of TNF and IL1B from both stimulated and nonstimulated cells were higher following 24 h of lying deprivation (d 0) compared with baseline (d -1). Abundance of IL6 was increased in nonstimulated cells after lying deprivation compared with baseline. In contrast, human disturbance for 24 h did not alter TNF, IL1B, or IL6 abundance relative to baseline levels. These results suggest that a short period of lying deprivation generally increases inflammatory responses but not metabolic responses.