A transient increase in cortical neuronal activity is followed by a complex process resulting in a transient increase in cerebral blood flow. This physiological process of neurovascular coupling, measured by its hemodynamic response function (HRF), is used to infer neuronal activity in modern vascular-based brain imaging techniques. Quantification of HRF during spontaneous brain activity remains, however, challenging. Neurovascular coupling is commonly assessed by averaging multiple neural and vascular responses triggered by visually or somatosensory stimuli. Here we tested whether HRF during spontaneous cerebral activity could be investigated by taking advantage of the whole-brain discontinuous EEG burst-suppression (BS) state during deep anesthesia. Experiments were carried out in adult male Wistar rats during BS induced by chloral hydrate overdose. The electro-corticographic (ECoG) and laser Doppler (LD) signals were simultaneously acquired from the left hemisphere. The rectified ECoG was convoluted with the HRF to obtain a synthetic LD signal. The HRF peak response latency was then programmatically optimized to obtain a maximum correlation between the recorded and the synthetic LD signals within the 0.1-0.4 Hz band. We found that with increasing depth of anesthesia, the decrease in the bursting frequency, was associated with an increase in HRF peak latency. Furthermore, this process was impaired following an ischemic brain injury. Our study provides proof of concept that HRF could be estimated during “spontaneous” BS opening a new non-invasive insight into assessing neurovascular coupling.