AbstractFoundations of photosynthesis research have been established mainly by studying the response of plants to changing light, typically to sudden exposure to a constant light intensity after a dark acclimation or to light flashes. This approach remains valid and powerful, but can be limited by requiring dark acclimation prior to time-domain measurements and often assumes that rate constants determining the photosynthetic response do not change between the dark- and light-acclimation.We present experimental data and a mathematical model demonstrating that these limits can be overcome by measuring plant responses to sinusoidally modulated light of varying frequency. By its nature, such frequency domain characterization is performed in light-acclimated plants with no need for prior dark acclimation. Amplitudes, phase shifts and upper harmonic modulation extracted from the data for a wide range of frequencies can target different kinetic domains and regulatory feedbacks. The occurrence of upper harmonic modulation reflects non-linear phenomena, including photosynthetic regulation. To support these claims, we present a frequency and time domain response in chlorophyll fluorescence emission of the green alga Chlorella sorokiniana in the frequency range 1000 – 0.001 Hz. Based on these experimental data and on numerical as well as analytical mathematical models, we propose that the frequency domain measurements can become a versatile new tool in plant sensing.One sentence summaryIt is proposed to characterize photosynthesis in the frequency domain without the need for dark adaptation and, thus, without assumptions about the dark-to-light transition.