Startle is a fast response elicited by sudden acoustic, tactile or visual stimuli in a variety of animal species and in humans. The magnitude of startle response can be modulated by external and internal variables and can be a useful tool to study the sensory-motor integration in animals. Different stimuli have been used to induce startle in horses, which makes it difficult to compare the responses to these different approaches. The present study uses ultra-short-term heart rate variability (HRV) analysis to characterize the cardiac autonomic modulation, reactivity assessment and blood cortisol measurements to describe the behavioural and endocrine responses to a simple, easy to replicate, effective and safe method of startle (an umbrella is abruptly opened near the horse). The ultra-short-term (64 s) heart rate (HR) series were interpolated (4 Hz) and divided into 256 points segments then the spectra calculated (Fast Fourier Transform). The spectra were then integrated into low (LF; 0.01–0.07 Hz; Index of Cardiac Sympathetic Modulation) and high (HF; 0.07–0.50 Hz; Index of Cardiac Parasympathetic Modulation) frequency bands. Following the startle test, the HR (p = 0.0101), the power of the LF band of the cardiac interval spectrum (p = 0.0002) and the LF/HF ratio (p = 0.0066) were found to be higher, whereas the power of the HF band of the cardiac interval spectrum was found to be lower (p = 0.0002). Also, the horses showed a noticeable escape response, with latency of reaction varying from 0.28 to 1.28 s, duration of reaction ranging from 1.52 to 7.92 s and escape distance covered varying from 3.43 to 9.97 m. However, the endocrine measurements failed to reveal significant changes in the cortisol levels after the startle test. We conclude that the startle test used in the current study was effective to produce changes in behavioural parameters and cardiac autonomic modulation of the horses and can therefore be an appropriate tool for neurobiological studies. Furthermore, the use of ultra-short segments (64 s) for HRV analysis appears to be effective and promising for the detection of mental stress in horses.