OBJECTIVES: To assess the influence of 2nd-degree AV blocks (AVB) on RR interval-based heart rate variability (HRV) variables; to investigate the effect of using PP interval time series and of artifact filtering on HRV analyses; to investigate the influence of electrocardiogram (ECG) recording length and time of recording; and to calculate day-to-day variability and reference intervals of HRV variables. ANIMALS: Thirty healthy adult horses. METHODS: RR and PP interval time series were extracted from 10-h Holter ECGs and an automated filter was applied to the RR time series (RR(f)). Time-domain HRV variables were calculated based on RR, PP, and RR(f) time series and their relation to the number of AVBs was assessed. Hourly 10- and 60-min segments were extracted to investigate the influence of segment length and recording time on HRV variables. Day-to-day variability and reference intervals of HRV variables were calculated. RESULTS: Variables of short-term HRV were significantly influenced by the number of AVBs when based on RR, but not when based on PP- and RR(f) time series. PP- and RR(f)-based HRV variables were in good agreement. The majority of HRV variables were influenced by recording time and ECG segment length. Day-to-day variability of HRV variables was low when based on 10-h ECG recordings but moderate to high when based on 60-min and 10-min recordings. CONCLUSIONS: Second-degree AVBs significantly influence conventional RR-based, but not PP- and RR(f)-based time-domain HRV variables. However, PP and RR(f) analyses have limitations and recording length and time of recording must be considered.