Background The handling of unknown weights, which is common in daily routines either at work or during leisure time, is suspected to be highly associated with the incidence of low back pain (LBP). Objectives To investigate the effects of knowledge and magnitude of a load (to be lifted) on brain responses, autonomic nervous activity, and trapezius and erector spinae muscle activity. Methods A randomized, within-subjects experiment involving manual lifting was conducted, wherein 10 participants lifted three different weights (1.1, 5, and 15 kg) under two conditions: either having or not having prior knowledge of the weight to be lifted. Results The results revealed that the lifting of unknown weights caused increased average heart rate and percentage of maximum voluntary contraction (%MVC) but decreased average inter-beat interval, very-low-frequency power, low-frequency power, and low-frequency/high-frequency ratio. Regardless of the weight magnitude, lifting of unknown weights was associated with smaller theta activities in the power spectrum density (PSD) of the central region, smaller alpha activities in the PSD of the frontal region, and smaller beta activities in the PSDs of both the frontal and central regions. Moreover, smaller alpha and beta activities in the PSD of the parietal region were associated only with lifting of unknown lightweights. Conclusions Uncertainty regarding the weight to be lifted could be considered as a stress-adding variable that may increase the required physical demand to be sustained during manual lifting tasks. The findings of this study stress the importance of eliminating uncertainty associated with handling unknown weights, such as in the cases of handling patients and dispatching luggage. This can be achieved through preliminary self-sensing of the load to be lifted, or the cautious disclosure of the actual weight of manually lifted objects, for example, through clear labeling and/or a coding system.