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The gastrointestinal (GI) tract plays a critical role in nutrition and the pathophysiology of disease, and there is an increasing variety of methodologies available for the assessment of various aspects of GI physiology. Advancements in assessment methods, including techniques to study gut motility, fermentation, permeability, and microbiota composition, have provided researchers with powerful tools to investigate the impact of diet on GI tract physiology and the microbiota-gut-brain axis. Mechanistic evidence from reverse translational studies, which apply findings from human studies to preclinical models in a "bedside-to-bench" approach, have also enhanced our understanding of the bidirectional interactions and candidate signalling molecules among the diet-gut-brain relationship. Interpreting data from these advanced techniques and study designs requires a thorough understanding of their principles, applications, and limitations. This review aims to summarise the methodological advances in GI tract physiology measurements and their application in nutritional studies, focusing on gut motility, fermentation, and permeability. We will present examples of how these techniques have been utilised in recent research, discuss their advantages and limitations, and provide insights on their use and interpretation in research. Understanding the capabilities and limitations of these tools is crucial for designing robust studies and elucidating the complex interplay between diet and the GI tract. The scope of this review encompasses recent advancements in GI tract assessment methodologies and their implications for nutritional research, providing a comprehensive overview for researchers in the field.