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Background: Plasma citrulline (CIT) concentration is considered to be a reliable marker of functional enterocyte mass, primarily in humans. However, information about CIT levels along with related metabolites, arginine (ARG), nitric oxide (NO), and ammonia in neonatal calves are lacking. Objectives: To compare plasma CIT, ARG, NO, and whole blood ammonia concentrations in neonatal calves with acute diarrhea with those in healthy calves and to assess their possible relationships with diarrhea‐related criteria. Animals: Seventy neonatal calves (60 with acute diarrhea and 10 healthy). Methods: Observational case‐control study. Diarrheic calves were classified into subgroups on the basis of etiology, severity of diarrhea, degree of dehydration, and systemic inflammatory response syndrome (SIRS) status. Plasma CIT and ARG concentrations were measured by liquid chromatography/tandem mass spectrometry. Results: Plasma CIT (median [range]: 67.5 [61.9‐75.4] vs 30.1 [15.0‐56.1] μmol/L) and ARG (170.7 [148.5‐219.5] vs 106.1 [54.4‐190.7] μmol/L) were lower and plasma NO (4.42 [3.29‐5.58] vs 6.78 [5.29‐8.92] μM) and blood ammonia concentrations (28.7 [26.1‐36.9] vs 59.8 [34.6‐99.5] μmol/L) were higher in the neonatal calves with diarrhea (P < .001). Plasma CIT (β = −0.29, P = .02), ARG (β = −0.33, P = .01), NO (β = 0.55, P < .001), and blood ammonia (β = 0.63, P <.001) were affected by SIRS status. Except for ammonia (0.52), the effects sizes for severity of diarrhea and degree of dehydration were small (ηp2 ≤ 0.45) for CIT, ARG, and NO. Conclusions and Clinical Importance: The changes in these variables might have diagnostic, prognostic, and therapeutic value in diarrheic neonatal calves. [ABSTRACT FROM AUTHOR]