The primary goal of this project was to demonstrate whether the relationship between bone mineral balance (BMB) and changes in the natural isotope composition of blood and urine observed in Earth-based bed rest studies could also be observed in crewmembers in spaceflight, providing the basis for inflight measurements of BMB and evaluation of effectiveness of bone loss countermeasures in individual crewmembers. We have achieved this goal. Although not all of our analyses are completed, analysis of archived urine samples from 32 crewmembers from previous ISS missions clearly shows the same pattern of change in Ca isotope composition observed in bed rest and predicted in spaceflight. More specifically, the 44Ca:42Ca ratio in urine (expressed as d44/42Ca) typically drops to below each crewmember’s individual average pre-flight value shortly after spaceflight begins, remains low during spaceflight, and returns to pre-flight values upon return to Earth. This is the pattern expected in crewmembers transitioning to more negative BMB in microgravity, and a confirmation of our ability to detect this change using Ca isotopes. A great deal more information can be extracted from the Ca isotope data we have gathered, a task that we have begun and will be our primary activity during the remainder of the project. Changes in Ca isotope composition are revealing details of the dynamics of BMB on previously inaccessible timescales, and offer new insights into bone biology. In particular, while on Ca isotopes show what on average crewmembers lose bone during spaceflight, our data reveal striking differences between the responses of individual crewmembers. Some of these differences are related to countermeasures. For example, crewmembers treated with both bisphosphonate and Advanced Resistive Exercise (ARED) uniformly showed not bone loss, while the response of crewmembers treated with exercise alone was more variable: most lost bone, some did not. Variations in response to treatment do not appear to be related to age, sex, or BMI (body mass index). The cause of these variations is of great interest, and we hope that further analysis will shed light on this and other questions. We do anticipate, however, that the answers to many questions will require more data from future projects. A secondary goal of our project was to increase the speed with which samples could be processed and analyzed. This goal is, of course, a moving target. Processing speed can always be increased, but we have made substantial improvements over where we were at the start of the project. We designed and validated an automated column chromatography procedure for faster and cleaner sample purification prior to analysis, and have validated a new microwave digestion system for improved sample digestion is now 80% complete. In addition, we are in the process of developing an improved mass spectrometric analytical procedure using a 46Ca-43Ca double spike, which should improve data quality, accuracy, and precision. Finally, we have begun exploring new technology to measure Ca isotope ratios using laser fluorescence rather than conventional mass spectrometry. Conventional mass spectrometers are not suited to either inflight or widespread clinical use. Such instruments are large, difficult or impossible to miniaturize, complex, and expensive, problems that could be overcome by using laser fluorescence. Whether a practical laser fluorescence device can be built remains to be seen, but preliminary results are promising, and efforts to build small and simple devices for measuring other isotopes (C and O) have been successful.