This study examined the characteristics of perceived discomfort and postural shifts at different magnitudes during a 4-h sitting period and the association between perceived discomfort and number of postural shifts. Forty healthy participants continuously typed a standardized text passage at a computer work station for 4 h. Subjects rated perceived body discomfort using Borg's CR-10 scale in 10 body regions (i.e. neck, shoulder, elbow, wrist/hand, upper back, lower back, buttock, thigh, knee, and ankle/foot). A seat pressure mat device was used to gather seat pressure data during sitting. Postural shifts were determined by analysis of the dispersion index of both ischial tuberosities from seat pressure data. The threshold for a postural shift was set at ±10% and ±20%. Perceived discomfort in all body regions increased continuously during a 4-h sitting period. The body regions with the highest perceived discomfort were the low back, buttocks, upper back, thigh, and neck. The average (±SD) numbers of postural shifts during the 1st, 2nd, 3rd, and 4th hour of sitting were 14.8 ± 9.5, 17.8 ± 9.4, 18.2 ± 11.1, and 18.1 ± 9.8 shifts per hour for the 10% threshold, and were 4.8 ± 4.4, 6.0 ± 5.6, 7.4 ± 6.7, and 7.7 ± 6.6 shifts per hour for the 20% threshold, respectively. Prolonged sitting led to an increase in perceived musculoskeletal discomfort over time. The number of postural shifts at both magnitudes increased in the first 2 h of sitting and, in the second 2-h period of sitting, only the number of larger postural shifts (with 20% threshold) increased. The findings extend our understanding of sitting behaviors.