Water filters and sediment trap samples were collected weekly from late May to early September 2009 from four lakes of the Lofoten archipelago, northwestern Norway, and were used to explore the applicability of the alkenone unsaturation index (UK37) for temperature reconstruction in limnic systems in the area. For the first time, we observed the occurrence of long-chain alkenones (LCAs) within the water columns of lakes in this region. Water filters from two of the four studied lakes contained measurable concentrations of alkenones that were restricted to spring turnover and disappeared with the onset of summer stratification. These results indicate that alkenones in the lake sediment of these lakes reflect biological production and temperature during lake mixing, taking place in late spring to early summer. Measurements from sediment trap material collected over the sampling season combined with water temperature measurements from automated data loggers provide an in situ calibration of the alkenone paleothermometer (Temperature = 33.0 x UK37 + 22.8; N=10; R2=0.95). Notably, this calibration reveals a UK37 sensitivity to temperature (i.e., the slope of the relationship) that is very similar to previous calibrations reported from both marine and lacustrine environments. LCAs can therefore serve as the first quantitative proxy for reconstructing past temperature variability from the Lofoten Islands. Based on this temperature calibration, a high-resolution temperature record was reconstructed over the past millennium, which shows unprecedented lake surface temperature warming during the past decades.