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1. Physical distance. You have likely noticed that downloading from a local server location is faster than a distant one. This is because the TCP window (e.g. HTTP transfers) is not optimized for the increased latency that comes from an increase in physical distance the bits have to travel. HTTP is bound by the TCP congestion window which determines how many packets can be sent at one time before an acknowledgment. The larger the window size, the higher the TCP throughput.
2. Firewalls, wireless, Local LAN, PC and modem issues. There are many localized issues which may negatively impact speedtest results. The best (but not perfect) PC test environment is to have a high end PC capable of delivering the provisioned speeds, directly attached to the modem.
3. Capacity issues with intermediate ISPs between the server and the user. There are some speedtest servers on ISPs with poor interconnect relationships or capacity issues. The path between the user and the server factors into any results. Local, on-net, speedtest servers are best to measure your provisioned speed.
4. Tuning of the Speedtest server with parallel TCP streams. This is not well known, but does have a direct impact to higher broadband speeds. Most speedtest servers open multiple TCP streams to more accurately account for single stream TCP limitations. How many simultaneous TCP streams a server opens is important as with 4 streams a server is accurate to up to 50Mbps testing on an average PC. Some speedtest servers are configured for even fewer streams and therefor are less accurate at higher speeds.
5. Asymmetric test environments - High end commercial connections with symmetric speeds often ask why they get asymmetric results from most speedtest servers. This is because most speedtest software / servers are configured for residential broadband testing. They are normally configured for 2-4 simultaneous download streams / test, but only 1-2 upload streams. This will inaccurately give results of asymmetric bandwidth even when you have symmetric speeds. Those with higher end work connections can simulate this.