Studies of the maximal oxygen uptake (VO(2max)) of transfemoral amputees have mostly used protocols that activate a relatively small muscle mass. Consequently, transfemoral amputee VO(2max) may be systematically underestimated, and the validity of these test protocols is questionable. (1) Investigate validity and reliability of a VO(2max) walking protocol and (2) compare the VO(2max) of a transfemoral amputee group with a group of matching controls. (1) Randomized crossover study: walking versus running VO(2max) for the control group and (2) case-control study: transfemoral amputees versus control group VO(2max). Twelve transfemoral amputees and control participants performed a walking VO(2max) test with increasing treadmill inclinations to voluntary exhaustion. The control group also completed a running ("gold-standard") VO(2max) test. Mean (standard deviation) control group VO(2max) following walking and running was similar, that is, 2.99 (0.6) L min(-1) and 3.09 (0.7) L min(-1), respectively. Mean (standard deviation) transfemoral amputee walking VO(2max) was 2.14 (0.8) L min(-1) (compared to CON; p < 0.01). Mean intraclass correlation coefficient of repeated VO(2) measurements was 0.97, and within-subjects standard deviation was 60 mL min(-1). The walk protocol is valid. Walking VO(2max) of transfemoral amputees was 40% lower compared to control group. Reliability of the walking protocol is comparable to other walking protocols. Clinical relevance The design, alignment, and materials of prostheses are important for effective ambulation. Cardio-respiratory fitness is, however, also important in this regard, and a low fitness may compromise health and independent living. Hence, transfemoral amputees with low physical fitness should engage in regular physical activity to improve health, gait capacity, and independency.