Pressure ulcer risk assessment might be optimized by incorporating the soft tissue reaction to mechanical loading in the currently used risk assessment scales. Cytokines, like IL-1alpha, IL-1RA, IL-8, and TNF-alpha, might be used to determine this tissue reaction, since they are released after 24 h of mechanical loading of epidermal equivalents. In the current study, the release and transport of these cytokines with time was evaluated. Epidermal equivalents were subjected to 20 kPa for different time periods (1, 2, 4, 6, 8, 16, and 24 h). Compared to the unloaded control group, a significant increase was found for IL-1alpha (4.7-fold), IL-1RA (4.8-fold), and IL-8 (3.6-fold) release after 1 h loading. For TNF-alpha, the release was significantly increased after 4 h of loading (5.1-fold compared to the unloaded situation), coinciding with the first signs of gross structural tissue damage. These cytokine values were determined in the surrounding medium and a transport model was developed to evaluate the distribution of cytokines inside the culture. These simulations revealed that all IL-8 and TNF-alpha was released from the keratinocytes, whereas most of the IL-1alpha and IL-1RA remained inside the keratinocytes during the 24 h loading period. In conclusion, IL-1alpha, IL-1RA, and IL-8 appear promising biochemical markers for pressure ulcer risk assessment, since their release is increased after 1 h of epidermal loading and before the onset of structural tissue damage.