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Neurovascular coupling reflects the close temporal and regional linkage between neural activity and cerebral blood flow. Although providing mechanistic insight, our understanding of neurovascular coupling is largely limited to nonphysiological ex vivo preparations and non-human models using sedatives/anesthetics with confounding cerebrovascular implications. Herein, with particular focus on humans, we review the present mechanistic understanding of neurovascular coupling and highlight current approaches to assess these responses and the application in health and disease. Moreover, we present new guidelines for standardizing the assessment of neurovascular coupling in humans. To improve the reliability of measurement and related interpretation, the utility of new automated software for neurovascular coupling is demonstrated, which provides the capacity for coalescing repetitive trials and time intervals into single contours and extracting numerous metrics (e.g., conductance and pulsatility, critical closing pressure, etc.) according to patterns of interest (e.g., peak/minimum response, time of response, etc.). This versatile software also permits the normalization of neurovascular coupling metrics to dynamic changes in arterial blood gases, potentially influencing the hyperemic response. It is hoped that these guidelines, combined with the newly developed and openly available software, will help to propel the understanding of neurovascular coupling in humans and also lead to improved clinical management of this critical physiological function.