Complex challenges arise when quantifying ecological processes over large temporal and spatial extents. To overcome these challenges, a complex spatiotemporal sampling design is required. The National Ecological Observatory Network (NEON) is a continental-scale observation facility designed to collect long-term, open access, ecological data to enable researchers to better understand how ecosystems in the United States are changing. The NEON sampling design enables the observatory to deliver high-quality, scalable data products through standardized and quality-controlled data collection methods. Data from NEON’s 34 aquatic sites (24 wadeable streams, 3 non-wadeable rivers, 7 lakes) span 19 ecoclimatic domains, and each site is 6+ years into its 30+ year lifetime. Coupling the standardized design with the large spatial extent, NEON aquatic sites offer open access data scalable from individual transects to the continental United States, and from individual measurements to a decade long period of record. Here, we introduce the spatiotemporal sampling design at NEON’s aquatic sites and present how NEON’s observational and instrumented data products can be integrated and scaled to address ecological questions across time and space including, but not limited to, questions such as: 1) how does aquatic macroinvertebrate community composition or surface water grab sample chemistry respond to changes in sensor-derived streamflow or water quality across NEON’s different ecoclimatic domains, or 2) can relationships between NEON sensor measurements and grab sample chemistry data be used to expand the temporal extent of ecologically-relevant analytes present in the grab samples (e.g., dissolved organic carbon, total suspended solids)? With >60 data products available from NEON’s aquatic sites covering a large proportion of the biological, physical, and chemical realms of a freshwater ecosystem, we aim to show that NEON data can be used to examine different dimensions of scale and can enable multiscale research in a findable, accessible, interoperable, and reusable way.