In recent years, injection of clove oil was investigated as potential alternative to the use of a hot iron for disbudding due to its cytotoxic and anaesthetic properties. Isoeugenol, the isomer of the main component of clove oil, is available as a pure substance and has some advantages as compared to clove oil, but its effects were studied rarely. In this paper we investigated behaviour, heart rate, heart rate variability and surface temperature around the horn bud of 40 calves in four treatments (N = 10/treatment): injection under each horn bud of 1.5 ml clove oil (CLOV), isoeugenol (ISO) or saline (CON), or hot-iron disbudding (BURN) with local anaesthesia and sedation. Behaviour, heart rate (HR) and heart rate variability (HRV) as well as surface temperature around the horn buds were measured before and at different time points after the treatment up to 7 days post treatment; behaviour was also observed during the treatment. Behavioural observations were performed real-time or per video recording. HR and HRV were analysed during undisturbed lying periods and post-treatment changes relative to baseline were calculated. The maximum surface temperature in the region around each of the two horn buds was used for further analysis. LMM, ANOVA or, for behaviour during treatment, non-parametric tests were used for statistical analysis. There was a treatment*time point interaction (LMM, p<0.05) for behaviours associated with pain (ear flicking, head shaking, head scratching) as well as for self-grooming. HR or HRV changes from baseline did not differ between treatments, but sample size was strongly reduced due to lack of data. The development of the maximum surface temperature around the horn buds differed clearly between treatments. Our findings suggest that the injection of clove oil and especially isoeugenol causes less pain compared to the use of a hot iron without analgesia, but more pain compared to the injection of saline solution.