Objectives Diaphragmatic dysfunction (DD) is often underdiagnosed in veterinary medicine. Various ultrasonographic diaphragmatic parameters, including diaphragmatic excursion (DE), diaphragmatic thickness (Tdi) and diaphragmatic thickening fraction (DTF), can be used to identify DD. We aimed to establish normal reference intervals in healthy cats, examine the effects of intrinsic factors on diaphragmatic parameters and evaluate the efficacy of these parameters in identifying DD in healthy and diseased cats. Methods This prospective observational study was conducted on 22 healthy cats and 12 diseased cats using ultrasonographic techniques to evaluate DE, Tdi and DTF. The correlation between diaphragmatic parameters and intrinsic factors, including sex, age, body weight and body condition score, was analysed using Pearson correlation analysis. Cut-off values for healthy and diseased cats were established using receiver operating characteristic curve analysis. Results The mean DEs of the left and right hemidiaphragms were 0.66 ± 0.16 and 0.64 ± 0.18 cm, respectively. The mean Tdis in the end-inspiration phase were 0.13 ± 0.03 and 0.12 ± 0.03 cm for the left and right hemidiaphragms, respectively, whereas in the end-expiration phase the mean Tdi was 0.07 ± 0.03 cm for both hemidiaphragms. The DTF ranges for the left and right hemidiaphragms were 23.90–122.1% and 38.80–107%, respectively. Intrinsic factors had no significant impact on DE, Tdi and DTF. The DE measurements for the left hemidiaphragm were more accurate for evaluating diaphragmatic function. To distinguish between healthy and diseased cats, the cut-off value, sensitivity and specificity were 0.458 cm (area under the curve [AUC] 0.846), 75.00% and 86.36%, respectively, for the left hemidiaphragm and 0.423 cm (AUC 0.704), 41.67% and 100%, respectively, for the right hemidiaphragm. Conclusions and relevance Normal reference values for DE, Tdi and DTF were established for healthy cats. DE, particularly in the left hemidiaphragm, obtained using routine ultrasonography, improves the efficacy of identifying DD and assists clinicians in the diagnosis and treatment of cats with cardiorespiratory disease.