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Non-nutritive sweeteners (NNS) are frequently used as substitutes for added sugars. NNS are difficult to study due to the inability to accurately measure the amounts individuals consume, as well as limitations in study design and methods, including reliance on observational study designs, the use of diet soda as a proxy of total NNS intake, and the grouping of NNS into a single category rather than studying NNS as individual products. New dietary assessment methods and improved study design and methods are needed to advance researchers' abilities to study NNS and their impact on consumers' health. The objectives of this dissertation were to 1) determine validity and reproducibility of a novel NNS food frequency questionnaire (NNS-FFQ), 2) develop methodology for an objective NNS urinary biomarker, 3) identify an appropriate carrier for NNS intake in studies, and 4) examine the literature on the relationship between NNS and weight-related outcomes based on study design and methods. Objectives 1 and 2: participants (n=125) completed three 24-hr dietary recalls, the NNS-FFQ, and 2 24-hr urine samples. NNS intake via NNS-FFQ and recalls were compared using Bland-Altman analyses, with agreement levels ranging from 92.7-99.2% for individual NNS types and total intake. The NNS biomarker methodology was developed using ultra performance liquid chromatography (UPLC-MS/MS), which analyzes each sample for the presence of NNS and related metabolites. This method observed a range from very strong presence of NNS to not detectable, indicating that this biomarker could identify specific NNS consumption (n=9). Objective 3: a sensory evaluation (n=67) was conducted to identify if applesauce or water was a more appropriate carrier for NNS for future interventions. Applesauce was preferred (sucralose=83.6%; aspartame=79.1%; stevia=74.6%) significantly more than water (p≤0.001), indicating that applesauce could be used as an acceptable carrier of NNS in research studies. Objective 4: a systematic literature review focusing on study design and methods used in investigations on NNS and weight-related outcomes found that 81% of RCT had improved weight outcomes, while 76% of observational studies had higher weight outcomes. Improving NNS study design and methods will increase the quality of research conducted on NNS and related health outcomes. / Ph. D. / Artificial sweeteners [non-nutritive sweeteners (NNS)] are often used to replace added sugars. NNS are difficult to study due to the inability to accurately measure the amounts individuals consume, as well as limitations in study design and methods, including reliance on observational study designs, the use of diet soda to represent total NNS intake, and the grouping of NNS into a single category rather than studying NNS as individual products. New dietary assessment tools and improved study design and methods are needed to allow researchers better to study NNS and their impact on health. The objectives of this dissertation were to 1) to determine the ability of a NNS food frequency questionnaire (NNS-FFQ) to measure typical NNS intake, 2) develop methodology for an objective NNS urinary biomarker, 3) identify an appropriate carrier for NNS intake in studies, and 4) to review the currently available research on the relationship between NNS and weight status. Objectives 1 and 2: 125 participants completed three 24-hr dietary recalls, the NNS-FFQ to measure usual NNS intake, and 24-hour urine samples for a NNS biomarker. Amounts of NNS that consumers reported in recalls were compared to amounts reported in the NNS-FFQ, with the tools finding similar NNS amounts in participants’ diets. The NNS biomarker methodology was developed using ultra performance liquid chromatography (UPLC-MS/MS). This rapid method measures the presence of NNS and related products (saccharin, acesulfame potassium, sucralose, steviol glucuronide, and erythritol) in urine. Among 9 participants, this method identified wide differences, ranging from strong presence of NNS to not measureable. Objective 3: participants (n=67) completed a sensory evaluation (taste testing) study to identify a more appropriate food or beverage (applesauce or water) for NNS (sucralose, aspartame, and stevia) to be mixed with in research studies. Applesauce was preferred over water for all sweeteners (>74%), indicating that applesauce would be an acceptable NNS carrier. Objective 4: a systematic review on study design and research methods used in studies on NNS and weight-related outcomes. Improving NNS measurement tools and study design methods will improve the quality of research that can be conducted on NNS and related health outcomes.