Limited information is available on injection forces of parenterals representing the in vivo situation. Scope of the present study was to investigate the contribution of the subcutaneous (sc) tissue layer to injection forces during in vivo injection. Göttingen minipigs received injections of isotonic dextran solutions (1–100 mPas) into the plica inguinalis using different injection rates and volumes (0.025–0.2 mL/s and 2.5 vs. 4.5 mL). The contribution of the sc back-pressure to injection forces was found to increase linearly with viscosity and injection rate ranging from 0.6 ± 0.5 N to 1.0 ± 0.4 N (1 mPas), 0.7 ± 0.2 N to 2.4 ± 1.9 N (10 mPas), and 1.8 ± 0.6 N to 4.7 ± 3.3 N (20 mPas) for injection rates of 0.025 to 0.2 mL/s, respectively. Variability increased with viscosity and injection rate. Values are average values from 10 randomized injections. A maximum of 12.9 N was reached for 20 mPas at 0.2 mL/s; 6.9 ± 0.3 N was determined for 100 mPas at 0.025 mL/s. No difference was found between injection volumes of 2.5 and 4.5 mL. The contribution of the tissue was differentiated from the contribution of the injection device and a local temperature effect. This effect was leading to warming of the (equilibrated) sample in the needle, therefore smaller injection forces than expected compensating tissue resistance to some parts. When estimating injection forces representative for the in vivo situation, the contribution of the tissue has to be considered as well as local warming of the sample in the needle during injection.