BACKGROUND: Autonomic modulation of heart rhythm is thought to influence the pathophysiology of myxomatous mitral valve disease (MMVD). HYPOTHESES: (1) Holter-derived variables reflecting autonomic modulation of heart rhythm change with MMVD severity in Cavalier King Charles Spaniels (CKCS); (2) Holter-derived variables can identify MMVD severity in CKCS; and (3) Holter-derived variables in CKCS in congestive heart failure (CHF) secondary to MMVD differ from those in dogs of other breeds in CHF. ANIMALS: Ninety privately owned dogs: 70 CKCS with variable MMVD severity and 20 non-CKCS in CHF secondary to MMVD. METHODS: Dogs were prospectively recruited and divided into 5 MMVD severity groups based on history, breed, and physical and echocardiographic examination findings. Holter-derived variables included heart rate variability (HRV), heart rate (HR), and arrhythmia evaluated from 24-hour Holter recordings. RESULTS: In CKCS, 18 of 26 HRV (all P < .0002) and 3 of 9 arrhythmia (all P < .0004) variables decreased with increasing MMVD, whereas minimum and mean HR (all P < .0001) increased with increasing MMVD severity. An arrhythmia variable representing sinus arrhythmia ("premature normals") (P < .0001) and the HRV variable triangular index (TI) (P < .0001) could distinguish CKCS with moderate or severe mitral regurgitation from CKCS in CHF in specific intervals. Among dogs in CHF, Holter-derived variables did not differ among breeds. CONCLUSIONS AND CLINICAL IMPORTANCE: In CKCS, Holter-derived variables changed with MMVD severity. "Premature normals" and TI showed diagnostic potential. Breed differences were not seen among dogs in CHF secondary to MMVD.