Background Biological variation helps determine whether population‐based or subject‐based reference intervals are more appropriate to assess changes in serial analytical values. Previous studies have investigated the biological variation of biochemical analytes weekly or with variable frequency over 5‐14 weeks in cats, but none have considered biological variation at less frequent intervals over 1 year. Objectives We aimed to evaluate the long‐term biological variation of 19 biochemical analytes in clinically healthy cats. Methods A prospective, observational study in which 15 clinically healthy, client‐owned cats were sampled for serum biochemical analyses every 8 weeks for 1 year. Frozen serum samples were single‐batch analyzed. Restricted maximum likelihood estimation was used to determine the coefficients of variation (CV), describing variation within each cat, between cats, and the analytical variation. These CVs were used to determine the indices of individuality and reference change values (RCVs). Results Albumin, alkaline phosphatase, creatine kinase, and globulin had high indices of individuality, indicating that they are best evaluated by RCVs. Phosphorus, potassium, chloride, sodium, symmetric dimethylarginine, and total CO2 had low indices of individuality, indicating that population‐based reference intervals are appropriate. Alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, calcium, cholesterol, creatinine, glucose, total bilirubin, and total protein had intermediate indices of individuality, indicating that RCVs may provide additional insight into the interpretation of analyte measurements beyond the population‐based reference intervals. Conclusions For many analytes, the biological variation detected was similar to that reported in prior studies. Clinicians should consider the biological variation of analytes to best interpret clinically relevant changes in serial analyte measurements.