An ultrasound indentation system with a pen-size hand-held probe was developed and used to obtain the effective Young's moduli of forearm and lower limb soft tissues in 12 subjects. Since the probe is manually driven, the alignment of the probe and control of the rate of indentation are parameters upon which the results obtained depend. This paper addresses whether manual indentation tests with the probe are sufficiently acceptable and repeatable for objective biomechanical characterization of limb tissues. Forearms of three normal subjects were tested in two states of muscular contraction. Six different indentation rates, ranging from 0.75 mm/s to 7.5 mm/s, were imposed. The load-indentation responses obtained were shown to be well represented by quadratic functions. A linear elastic indentation solution was used to extract the effective Young's modulus. The material parameters extracted were repeatable and rather rate-insensitive for the range of rates used. The effective Young's modulus obtained was found to significantly increase as a result of contraction of the underlying muscles. Indentor misalignment experiments demonstrated that misalignment affects the measurement from which the effective Young's modulus of soft tissues is calculated. This effect, however, was found to decrease as the tissue thickness increased. With the investigation of the above issues, a procedure has been established for the extraction of effective Young's moduli of limb soft tissues from manual cyclic indentation responses. Tests on experimental subjects' lower limbs further demonstrated that the ultrasonic indentor is a feasible instrument for characterization of the biomechanical properties of limb soft tissues. Paired-t tests showed that the effective Young's moduli of the lower limb soft tissues of three elderly persons with transtibial amputation were significantly smaller than those of six unimpaired young subjects.