BACKGROUND: Global Health Initiatives (GHIs), aiming at reducing the impact of specific diseases such as Human Immunodeficiency Virus (HIV), have flourished since 2000. Amongst these, PEPFAR and GFATM have provided a substantial amount of funding to countries affected by HIV, predominantly for delivery of antiretroviral therapy (ARV) and prevention strategies. Since the need for additional human resources for health (HRH) was not initially considered by GHIs, countries, to allow ARV scale-up, implemented short-term HRH strategies, adapted to GHI-funding conditionality. Such strategies differed from one country to another and slowly evolved to long-term HRH policies. The processes and content of HRH policy shifts in 5 countries in Sub-Saharan Africa were examined. METHODS: A multi-country study was conducted from 2007 to 2011 in 5 countries (Angola, Burundi, Lesotho, Mozambique and South Africa), to assess the impact of GHIs on the health system, using a mixed methods design. This paper focuses on the impact of GFATM and PEPFAR on HRH policies. Qualitative data consisted of semi-structured interviews undertaken at national and sub-national levels and analysis of secondary data from national reports. Data were analysed in order to extract countries' responses to HRH challenges posed by implementation of HIV-related activities. Common themes across the 5 countries were selected and compared in light of each country context. RESULTS: In all countries successful ARV roll-out was observed, despite HRH shortages. This was a result of mostly short-term emergency response by GHI-funded Non-Governmental Organizations (NGOs) and to a lesser extent by governments, consisting of using and increasing available HRH for HIV tasks. As challenges and limits of short-term HRH strategies were revealed and HIV became a chronic disease, the 5 countries slowly implemented mid to long-term HRH strategies, such as formalisation of pilot initiatives, increase in HRH production and mitigation of internal migration of HRH, sometimes in collaboration with GHIs. CONCLUSION: Sustainable HRH strengthening is a complex process, depending mostly on HRH production and retention factors, these factors being country-specific. GHIs could assist in these strategies, provided that they are flexible enough to incorporate country-specific needs in terms of funding, that they coordinate at global-level and minimise conditionality for countries.