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Abstract Understanding the relationship between intraspecific trait variability (ITV) and its biotic and abiotic drivers is crucial for advancing population and community ecology. Despite its importance, there is a lack of guidance on how to effectively sample ITV and reduce bias in the resulting inferences. In this study, we explored how sample size affects the estimation of population‐level ITV, and how the distribution of sample sizes along an environmental gradient (i.e., sampling design) impacts the probabilities of committing Type I and II errors. We investigated Type I and II error probabilities using four simulated scenarios which varied sampling design and the strength of the ITV‐environment relationships. We also applied simulation scenarios to empirical data on populations of the small mammal,Peromyscus maniculatusacross gradients of latitude and temperature at sites in the National Ecological Observatory Network (NEON) in the continental United States. We found that larger sample sizes reduce error rates in the estimation of population‐level ITV for both in silico andPeromyscus maniculatuspopulations. Furthermore, the influence of sample size on detecting ITV‐environment relationships depends on how sample sizes and population‐level ITV are distributed along environmental gradients. High correlations between sample size and the environment result in greater Type I error, while weak ITV–environmental gradient relationships showed high Type II error probabilities. Therefore, having large sample sizes that are even across populations is the most robust sampling design for studying ITV‐environment relationships. These findings shed light on the complex interplay among sample size, sampling design, ITV, and environmental gradients. 

Abstract Intraspecific trait variation (ITV) is an increasingly important aspect of biodiversity and can provide a more complete perspective on how abiotic and biotic processes affect individuals, species' niches and ultimately community‐level structure than traditional uses of trait means. Body size serves as a proxy for a suite of traits that govern species' niches. Distributions of co‐occurring species body sizes can inform niche overlap, relate to species richness and uncover mechanistic drivers of diversity.We leveraged individual‐level body size (length) in freshwater fishes and environmental data from the National Ecological Observatory Network (NEON) for 17 lakes and streams in the contiguous United States to explore how abiotic and biotic factors influence fish species richness and trait distributions of body size. We calculated key abiotic (climate, productivity, land use) and biotic (phylogenetic diversity, trait diversity, community‐level overlap of trait probability densities) variables for each site to test hypotheses about drivers of ITV in body size and fish diversity.Abiotic variables were consistently important in explaining variation in fish body size and species richness across sites. In particular, productivity (as chlorophyll) was a key variable in explaining variation in body size trait richness, evenness and divergence, as well as species richness.This study yields new insights into continental‐scale patterns of freshwater fishes, possible only by leveraging the paired high frequency, in situ abiotic data and individual‐level traits collected by NEON.