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Background Assessment of cardiac electromechanical function in horses requires training, experience and specialised equipment and does not allow continuous monitoring over time. Objectives The objective of this study was to establish the use of an acoustic ECG monitor (Audicor® ) in healthy horses. It provides noninvasive, examiner-independent, continuous analyses combining ECG and phonocardiography to calculate indices of cardiac mechanical activity and haemodynamics. Device usability was investigated, reference intervals calculated and reproducibility of analyses assessed. Study design Prospective descriptive study. Methods Continuous overnight recordings were obtained in 123 healthy horses. ECG and acoustic cardiography analyses were performed. Electromechanical activating time (EMAT), rate-corrected EMATc, left ventricular systolic time (LVST), rate-corrected LVSTc and intensity and persistence of the third and fourth heart sound (S3, S4) were reported. Associations with age and reproducibility of analyses were assessed. Results Audicor® recordings of diagnostic quality were obtained in 116 horses, with an artefact-free recording time of 1:08-14:03 h (mean 10:21 h). 44.8% of the horses had atrial premature complexes (up to 0.18% of analysed beats), 4.3% had ventricular premature complexes (up to 0.021% of analysed beats). Reference intervals for acoustic cardiography variables were reported. S3 was significantly more often graded ≥5 (scale 0-10) in younger compared to older horses (P = 0.0036, R2 = 0.072). The between-day coefficient of variation ranged from 2.5 to 7.7% for EMAT, EMATc, LVST and LVSTc. Main limitations Audicor® algorithms are based on human databases. Horses were deemed clinically healthy without advanced diagnostics. Some data were lost because of technical difficulties, artefacts and noises. Conclusions Overnight Audicor® recordings are feasible in horses. Combining ambulatory ECG and phonocardiography allows noninvasive, continuous assessment of variables representing systolic and diastolic cardiac function. ECG rhythm analyses require over-reading by a specialist, but acoustic cardiography variables are based on automated algorithms independent of examiner input. Further studies are required to establish the clinical value of acoustic cardiography in horses.