We studied biochemical changes in biofilm and suspended particulate and dissolved organic matter (OM) during the leaf emergence period (MarchâMay 2008) in a forested headwater stream in response to a long-term (4 years, 2004â2008) experimental nutrient enrichment study. This study compared results from one reach upstream of the enrichment point and one reach downstream using moderate nutrient concentrations (nitrogen, N, from 388 to 765 ÎŒg Lâ1 and phosphorus, P, from 10 to 30 ÎŒg Lâ1, resulting in N:P ratios of 85â56). During the spring of 2008, we analysed the chlorophyll content, elemental composition (carbon, C, and N), bacterial density, and extracellular enzyme activities along with their biochemical composition (amino acids, fatty acids and sterols) on biofilm and OM. Nutrients caused changes in the biochemical composition of the biofilm, while changes in the OM were subtle. The C:N ratio of the biofilm decreased with nutrient enrichment likely due to the increase in protein (non-essential amino acids). The polysaccharide and total and essential fatty acid contents were higher when nutrient enrichment coincided with greater light availability. The peptidase extracellular activity was higher in the fertilised reach at early spring, while phosphatase activity decreased at late spring. The suspended and dissolved OM composition did not change due to the nutrient addition, likely due to the lower water residence time in the reach. Headwater systems are highly dynamic, and the biochemical composition of the biofilm changed in response to changes in nutrients but also to light in this study. These changes, although moderate, could influence higher trophic levels through modifications in their diet. This experiment exemplifies how small land use shifts may affect headwater streamsThis work has been funded by the Spanish Ministry of Economy and Competitiveness with projects CGL2014-58760-C3-