Living cells constitute an extraordinary state of matter since they are inherently out of thermal equilibrium due to internal metabolic processes. Indeed, measurements of particle motion in the cytoplasm of animal cells have revealed clear signatures of nonthermal fluctuations superposed on passive thermal motion. However, it has been difficult to pinpoint the exact molecular origin of this activity. Here, we employ time-resolved microrheology based on particle tracking to measure nonequilibrium fluctuations produced by myosin motor proteins in a minimal model system composed of purified actin filaments and myosin motors. We show that the motors generate spatially heterogeneous contractile fluctuations, which become less frequent with time as a consequence of motor-driven network remodeling. We analyze the particle tracking data on different length scales, combining particle image velocimetry, an ensemble analysis of the particle trajectories, and finally a kymograph analysis of individual particle trajectories to quantify the length and time scales associated with active particle displacements. All analyses show clear signatures of nonequilibrium activity: the particles exhibit random motion with an enhanced amplitude compared to passive samples, and they exhibit sporadic contractile fluctuations with ballistic motion over large (up to 30 μm) distances. This nonequilibrium activity diminishes with sample age, 3 Equal contribution. New J. Phys. 16 (2014) 075010 M Soares e Silva et al dynamics in minimal cytoskeletal model systems. Active (nonthermal) fluctuations have been observed both for actin networks activated by skeletal muscle myosin II motors [32-36] and in one recent study for nematic microtubule solutions activated by kinesins [37]. Myosin II is a motor protein with two heads that bind to actin filaments and uses chemical energy derived from hydrolysis of adenosine triphosphate (ATP) to generate pN-forces [38]. Individual myosin motors are non-processive, with a duty ratio of only a few per cent at saturating ATP levels. Nevertheless, single-headed myosin II subfragments have been shown to increase the effective temperature of actin networks [39]. Bipolar myosin II filaments, which resemble myosin minifilaments in cells, are more processive and can cause actin network contraction [40][41][42][43]. A combined active and passive microrheology study revealed clear violations of the FDT at low frequencies for active actin-myosin networks [33,34]. Passive microrheology of these networks using video particle tracking revealed clear signatures of nonequilibrium activity in the particle fluctuations. Specifically, the distribution of displacements (or Van Hove correlations) showed a markedly non-Gaussian shape for spherical as well as rod-shaped probe particles [35,36]. In spatially homogeneous networks, non-Gaussianity is indeed a signature of nonthermal activity [44,45]. In crosslinked networks, myosin contractility can generate substantial contractile tension, which can stiffen actin networks by up to 100-fo...