The purposes of this study were to investigate the muscle-tendon unit stiffness response and to compare the stiffness with those of other indirect markers induced by two bouts of unaccustomed eccentric exercise. Eleven untrained men performed two bouts of 200 maximal eccentric contractions of the right quadriceps 4 weeks apart. Changes in stiffness, pain evoked by stretching and pressure, plasma creatine kinase (CK) activity, and muscle thickness were followed for 7 days after each bout. Stiffness and pain peaked immediately and 1 day after the first exercise bout, whereas CK and thickness were highest 4 and 7 days after the first exercise bout, respectively (p < 0.05 for all). Muscular pain, thickness, and stiffness responses were lower by 53.3%, 99%, and 11.6%, respectively, after the repeated bout compared to after the first bout (p < 0.05 for all), while CK activity response did not differ significantly between bouts. High responders for an increase in muscle-tendon unit stiffness showed a repeated-bout effect for stiffness, pain, and CK activity (by 29%, 65%, and 98%, p < 0.05 for all), but the repeated-bout effect was not that clear in low responders. These findings suggest that a repeated eccentric exercise bout effect on stiffness in quadriceps is mostly not associated with muscle pain and CK activity, but there are large individual differences.