BACKGROUND: Monitoring serum phosphorus concentrations is crucial in the management of chronic kidney disease in cats. The diurnal variation of serum phosphorus concentration may affect clinical assessment. HYPOTHESIS/OBJECTIVES: Characterize the diurnal variation of serum phosphorus concentration in cats and determine the associations between changes in serum phosphorus concentration and several metabolites of phosphorus metabolism. ANIMALS: Six apparently healthy, intact male, specific-pathogen-free cats were housed in a photoperiod, humidity, and temperature-controlled facility. METHODS: Blood sampling was performed hourly for 24 hours to obtain the serum concentrations of phosphorus, ionized calcium, parathyroid hormone, and calcidiol. Results were analyzed using linear mixed-effect models to determine the significance of diurnal variation and associations between serum phosphorus concentrations and other metabolites over time. RESULTS: Diurnal variation in serum phosphorus concentration was noted with an apex around 11:00 followed by gradually declining concentrations to reach the nadir around 23:00. The serum phosphorus concentration again increased through the early morning on the next day. An approximately 25% difference in serum phosphorus concentration at the apex and the nadir was documented. A non-linear relationship between the serum concentrations of phosphorus and ionized calcium over time was identified. CONCLUSIONS AND CLINICAL IMPORTANCE: Diurnal variations of serum phosphorus concentration and associations between the trending of serum phosphorus and ionized calcium concentrations were evident in a group of clinically healthy adult cats housed in a controlled environment. These findings can help in the interpretation of clinical and research data regarding calcium and phosphorus metabolism and kidney health in cats.