The hydrogen isotope composition (δ<sup>2</sup>H) of biomarkers produced by algae is strongly influenced by the δ<sup>2</sup>H values of the water in which they grew. δ<sup>2</sup>H values of algal biomarkers preserved in lake sediments are thus a useful tool for reconstructing past changes in lake water δ2H values, which can be used to infer hydroclimate variability. However, studies from laboratory cultures of marine algae have shown that a number of factors can influence the magnitude of hydrogen isotope fractionation between algal lipids and their source water, including temperature and growth rates. Quantifying the natural extent of these changes in freshwater lacustrine settings and identifying their causes is essential for robust application of δ<sup>2</sup>H values of algal lipids as paleohydroclimate proxies, yet the influence of these factors remains poorly constrained.<br><br> This work targets the effect of temperature and productivity on <sup>2</sup>H/<sup>1</sup>H fractionation in algal biomarkers through a comparative time series in two central Swiss lakes: eutrophic Lake Greifen and oligotrophic Lake Lucerne. Particulate organic matter was collected from surface waters at six time points throughout the spring and summer of 2015, and δ<sup>2</sup>H values of short chain fatty acids, as well as the diatom biomarker brassicasterol, were measured. We paired these measurements with in situ incubations conducted with NaH<sup>13</sup>CO<sub>3</sub>, which were used to calculate the production rates of individual lipids in lake surface water. As algal productivity increased from April to June, the magnitude of <sup>2</sup>H/<sup>1</sup>H fractionation in Lake Greifen increased by as much as 148 ‰ for individual fatty acids. During the same time period in Lake Lucerne, the magnitude of <sup>2</sup>H/<sup>1</sup>H fractionation increased by as much as 58 ‰ for individual fatty acids, consistent with the 2–4 ‰ per °C increase in <sup>2</sup>H/<sup>1</sup>H fractionation observed in cultures of microalgae. Larger changes in <sup>2</sup>H/<sup>1</sup>H fractionation in Lake Greifen may be due to a combined effect of higher temperatures and increased algal productivity, which can cause relatively greater contributions of highly depleted H from NADPH in Photosystem I to be incorporated into lipids, or due to seasonal changes in the structure of the algal community. Fatty acid δ<sup>2</sup>H values were preserved in surface sediment, while those of brassicasterol indicated large (~ 40 ‰) isotopic effects due to degradation. The magnitude of brassicasterol fractionation was significantly different between the two lakes, suggesting that its hydrogen isotope composition may be more sensitive to nutrient regime than that of fatty acids.