Protium-deuterium isotope separation and tritium enrichment experiments have been carried out under the condition of a total reflux cycle using a continuous twin-bed hydrogen isotope separation technique, i.e., a twin-bed periodically counter-current flow technique. Two beds were packed with Pd and LaNi4.7Al0.3, which show positive and inverse isotope effects, respectively. The separation efficiency was studied experimentally in terms of stoichiometry between hydrogen and adsorbents, cycles, and extraction ratio. The experimental results show that a steady distribution of hydrogen isotopes along the axial direction can be obtained within an operating period of three cycles and a 10% extraction ratio at a moderate H/Pd atomic ratio. The results of a tritium enrichment experiment carried out under optimized conditions indicate that good enrichment efficiency is possible using this method to separate a three-component gas when the extraction ratio is kept small. Since the column used in this experiment is relatively short, there is great potential for this method for meeting the requirements of large-scale operations if long columns or multi-bed combined systems are employed.