Terrestrial run-off is increasing in temperate lakes due to climate change and can lead to loading of colored dissolved organic matter (cDOM) and nutrients, thus reducing light availability and increasing carbon, nitrogen, and phosphorus. Run-off events are highly irregular, resulting in temporal resource variability that may determine the energy flow in planktonic communities. To understand the effects of run-off variability on natural plankton communities, we conducted a mesocosm experiment at SITES AquaNet in Lake Erken, Sweden. Treated mesocosms received equal total amount of cDOM and nutrients but at different frequencies and magnitudes (Daily, Intermittent, Extreme), while keeping an untreated Control. Here, we performed three surrogate prey incubation experiments with fluorescently labeled bacteria in the mesocosms to study the trophic strategies of nanoflagellates under the run-off scenarios. Our results show that phototrophic nanoflagellates increased under Daily and Intermittent additions of cDOM and nutrients at early stages but declined thereafter, likely due to light limitation and grazing by rotifers. Heterotrophic nanoflagellate biovolume was highest in the beginning, while the grazing rate on bacteria was highest in the middle of the experiment when bacterial abundance was highest. The mixotrophic nanoflagellate abundance was generally low and unaffected by the treatments, despite high bacterial densities and reduced light, while the highest abundance was found in the Control. The overall development of nanoflagellates was modulated by microzooplankton grazing pressure over time. Our study contributes to better understanding the influence of future global change, including variable terrestrial run-off scenarios, on food-web interactions considering both bottom-up and top-down processes.