Objectives: The aim of the study was to assess physiological tremor as a result of fatigue related to manual activities, taking into account age, gender and upper limb (dominant and non-dominant). Material and Methods: The study included participants from 2 age groups, 25–35 years and 55–65 years. Work fatigue was induced during a 3-hour (180 min) 2-handed upper limb control task which consisted of 3 stages. The first stage lasted 30 min, the second stage – 60 min (90 min from the beginning of the effort) and the third stage – 90 min (180 min from the beginning of the effort). Measurements of physiological tremor were performed before exercise and at 30 min, 90 min and 180 min of exercise duration. An acceleration measurement set-up for both upper limbs was used to collect tremor time courses. The power spectral density functions of the tremor signals were analyzed. Results: Amplitude indicators from the frequency range 1–5 Hz and 8–14 Hz were extracted for analysis. The age of the subjects did not significantly affect the parameters of physiological tremor. Amplitude indicators from lower (1–5 Hz) and higher (8–14 Hz) frequencies take lower average values for women. For both indicators, a significant difference was observed in the subsequent measurements. The mean values of both indicators for the non-dominant limb were significantly higher. Conclusions: Physiological tremor measurement carried out simultaneously for both upper limbs to analyze muscle fatigue during repetitive work can improve the efficiency of assessing the occurrence of fatigue on the job and increase the efficiency of assessing adaptive capabilities. Int J Occup Med Environ Health. 2026;39(1)