Background: It has long been contested that trade rules and agreements are used to dispute regulations aimed at preventing noncommunicable diseases (NCDs). Yet most analyses of trade rules and agreements focus on trade disputes, potentially overlooking how a challenge to a regulation’s consistency with trade rules may lead to ‘policy or regulatory chill’ effects whereby countries delay, alter, or repeal regulations in order to avoid the costs of a dispute. Systematic empirical analysis of this pathway to impact was previously prevented by a dearth of systematically coded data. Methods and findings: Here, we analyse a newly created dataset of trade challenges about food, beverage, and tobacco regulations among 122 World Trade Organization (WTO) members from January 1, 1995 to December 31, 2016. We thematically describe the scope and frequency of trade challenges, analyse economic asymmetries between countries raising and defending them, and summarise 4 cases of their possible influence. Between 1995 and 2016, 93 food, beverage, and tobacco regulations were challenged at the WTO. ‘Unnecessary’ trade costs were the focus of 16.4% of the challenges. Only one (1.1%) challenge remained unresolved and escalated to a trade dispute. Thirty-nine (41.9%) challenges focussed on labelling regulations, and 18 (19.4%) focussed on quality standards and restrictions on certain products like processed meats and cigarette flavourings. High-income countries raised 77.4% (n = 72) of all challenges raised against low- and lower-middle–income countries. We further identified 4 cases in Indonesia, Chile, Colombia, and Saudi Arabia in which challenges were associated with changes to food and beverage regulations. Data limitations precluded a comprehensive evaluation of policy impact and challenge validity. Conclusions: Policy makers appear to face significant pressure to design food, beverage, and tobacco regulations that other countries will deem consistent with trade rules. Trade-related influence on public health policy is likely to be understated by analyses limited to formal trade disputes.