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Understanding the key factors influencing the water quality of large river systems forms an important basis for the assessment and protection of cross-regional ecosystems and the implementation of adapted water management concepts. However, identifying these factors requires in-depth comprehension of the unique environmental systems, which can only be achieved by detailed water quality monitoring. Within the scope of the joint science and sports event “Elbschwimmstaffel” (swimming relay on the river Elbe) in June/July 2017 organized by the German Ministry of Education and Research, water quality data were acquired along a 550 km long stretch of the Elbe River in Germany. During the survey, eight physiochemical water quality parameters were recorded in high spatial and temporal resolution with the BIOFISH multisensor system. Multivariate statistical methods were applied to identify and delineate processes influencing the water quality. The BIOFISH dataset revealed that phytoplankton activity has a major impact on the water quality of the Elbe River in the summer months. The results suggest that phytoplankton biomass constitutes a substantial proportion of the suspended particles and that photosynthetic activity of phytoplankton is closely related to significant temporal changes in pH and oxygen saturation. An evaluation of the BIOFISH data based on the combination of statistical analysis with weather and discharge data shows that the hydrological and meteorological history of the sampled water body was the main driver of phytoplankton dynamics. This study demonstrates the capacity of longitudinal river surveys with the BIOFISH or similar systems for water quality assessment, the identification of pollution sources and their utilization for online in situ monitoring of rivers.