Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention towards a better understanding of microgravity, hypogravity (<1g) or hypergravity (>1g) induced alterations. In the present work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15 and 20g for 4 or 16 h, using a large diameter centrifuge (LDC). Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10 and 15g for 16 h, in comparison to 1g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency towards an up-regulation of tenogenic markers was observed; SCX, TNC, COL3A1 and DCN were significantly up-regulated in hTDCs cultured at 15g and COL3A1 and DCN were significantly up-regulated in hTDCs cultured at 20g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation.